JP2007531514A - Bioluminescence imaging of local Ca2 + dynamics in living organisms in non-invasive real time in vivo - Google Patents

Abstract

The present invention relates to an optical detection method of the Ca 2 ⁺ dynamics in biological systems, said method is present in said biological system, comprises or consists of chemiluminescent protein linked to a fluorescent protein, recombinant Ca ²⁺ sensitive poly Monitoring the photons emitted by the peptide. In certain embodiments, the recombinant polypeptide comprises or consists of aequorin and GFP linked by a linker that allows chemiluminescence resonance energy transfer (CRET), wherein the recombinant polypeptide is bound to a particular cellular domain. Or optionally includes peptide fragments that can be targeted to the compartment. The invention also relates to a transgenic non-human animal expressing the recombinant polypeptide sensitive to calcium concentration in conditions that allow monitoring of Ca ²⁺ kinetics in vivo. In certain embodiments, the expression and / or localization of the recombinant polypeptide is confined to a particular tissue, single cell type, and / or a particular cell compartment or domain.

Description

The present invention provides a method that allows optical detection of local calcium signaling (eg, Ca ²⁺ concentrated microdomains) using genetically encoded bioluminescent reporters. The present invention describes a method for detecting the effects of pharmacological or neuromodulating agents on local Ca ²⁺ signaling. The present invention relates specifically to cellular or tissue activation, such as nerve activation, and dynamic variation of local Ca ²⁺ with respect to optical detection of ion channel function (Ca ²⁺ permeable receptors / channels) and synaptic transmission. Provides a method for visualizing The invention also relates to a method for optical detection of Ca ²⁺ kinetics in a biological system, the method comprising a recombinant Ca ²⁺ sensitive polypeptide comprising or consisting of a chemiluminescent protein linked to a fluorescent protein present in the biological system. Monitoring the photons emitted by. The present invention also provides a trans-expressing recombinant polypeptide sensitive to calcium concentration, comprising at least one chemiluminescent protein linked to a fluorescent protein under conditions that allow in vivo monitoring of local calcium dynamics. A transgenic non-human animal is provided. Ca ²⁺ plays a role in almost all cell functions including fertilization, secretion, contraction-relaxation, cell motility, protein metabolism, synthesis, production, gene expression, cell cycle progression, and apoptosis in the cytoplasm and mitochondria. It is one of the most ubiquitous and physiologically important signaling molecules (Rizzuto et al., 2002).

The characteristics of Ca ²⁺ transients at the cellular and subcellular level are complex and vary with spatial, temporal and quantitative factors. There is a 20,000-fold difference or less in Ca ²⁺ concentration in the cytoplasm and extracellular space, so Ca ²⁺ inflow will occur at high speed even if the channel is open for only a short time. Factors such as diffusion, Ca ²⁺ binding to buffer proteins, and sequestration by cellular compartments produce Ca ²⁺ gradients that produce high concentrations of microdomains within a few hundred nm from the channel pores. Beyond longer distances such as 10 μm, the effective diffusion coefficient of Ca ²⁺ will decrease strongly.

The Ca ²⁺ signal has a defined profile (eg, amplitude and response rate) because space, time and amplitude are highly regulated. Ca ²⁺ transients (-transient) is, ^{Ca 2+} diffusion in the cytosol, is shaped by the buffering, and ^{Ca 2+} transport by organelles by ^{Ca 2+} binding proteins (Bauer, 2001; Llinas et al., 1995). The Ca ²⁺ concentration reached in any given cellular microdomain and its kinetics are very important in determining whether a signaling pathway has succeeded in reaching its target. Ca ²⁺ is required for the activation of many important cellular proteins, including enzymes such as kinases and phosphatases, transcription factors, and protein machinery involved in secretion. The Ca ²⁺ signaling cascade may also mediate negative feedback in the regulation of biochemical pathways or functional receptors and transport mechanisms. Intracellular Ca ²⁺ propagation also helps link local signaling pathways with more distant signaling pathways within the cell, or lengthens between cells or cellular networks (eg, the central nervous system). Distance transmission can be facilitated (Augustine et al., 2003).

Ca ²⁺ transients that produce high concentrations of Ca ²⁺ microdomains are a wide variety of functions that are important in development, secretion, and apoptosis, as well as many cellular processes, including gene expression, neurotransmission, synaptic plasticity, and neuronal cell death (Augustine et al., 2003; Bauer, 2001; Llinas et al., 1995; Neher, 1998). Characterizing the spatiotemporal specificity of the Ca ²⁺ profile is an early event associated with the development of many pathological processes (migraine, schizophrenia, and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease) Is important to elucidate the mechanisms responsible for intracellular Ca ²⁺ homeostatic disturbances (Mattson and Chan, 2003). Since Ca ²⁺ is directly or indirectly associated with almost all cell signaling pathways, optical detection of Ca ²⁺ is a versatile bioactivity at the molecular, cellular, tissue, and whole animal level. It is an indicator. Imaging of local Ca ²⁺ events using light microscopy has made great progress. As a result, Ca ²⁺ signaling was achieved in single dendritic spines (Yuste, 2003) and more recently in single synapses (Digregorio, 2003) using fluorescent dyes. However, one way to spatially improve the measurement of Ca ²⁺ is to genetically target the reporter protein to a specific location, which allows direct visualization of Ca ²⁺ activity. In particular, such reporter proteins could be anchored to the microdomain (within 200 nm from the source or receptor) or even into the nanodomain (within 20 nm) (for review see Augustine et al., 2003). Reporter gene expression under cell type-specific promoter control in transgenic animals is also non-invasive to track dynamic changes in imaging single cell types, tissues, or whole anatomical animals Specific methods can be provided.

Real-time calcium monitoring helps to improve the elucidation of the development, plasticity and function of biological systems such as the central nervous system. In fact, great efforts have been made to develop optical techniques for imaging electrical activity in single cell types, especially single neurons and neuron networks, but this goal has also been achieved using electrophysiological techniques. There is still a need to achieve. Gene targeting of Ca ²⁺ reporter probes in spatially confined regions of cells or biological systems (eg, within compartments, to microdomains or nanodomains, or by fusion to specific polypeptides) can affect specific cellular activity. Alternatively, a molecular imaging technique for detecting physiological functions. The present invention relates to a method (method for detecting the dynamics of Ca ²⁺ optically in a biological system is present in said biological system, comprises or consists of chemiluminescent protein linked to a fluorescent protein, recombinant Ca ²⁺ sensitive Such need in the art by providing a non-human animal expressing the recombinant polypeptide sensitive to calcium, as well as monitoring photons emitted by the polypeptide) To help satisfy. The non-invasive nature of this technology, as well as the finding that the recombinant protein is non-toxic, means that the method can probably be applied in humans.

  The invention will now be described with reference to the following drawings.

DESCRIPTION OF THE INVENTION Bioluminescent species are present in coelenterates. Numerous studies have shown that bioluminescence is generated by photoproteins that are often sensitive to calcium. Sensitivity as used herein when referring to a protein refers to any modification of the conformation of the sensitive protein, its affinity for other molecules, localization, or light emission. Certain proteins of this type emit a momentary light in response to an increase in calcium ion concentration. Of these photoproteins, Aequorin is one of the best studied (Blinks et al., 1976).

Aequorin isolated in the jellyfish Aequoria victoria (Shimomura et al., 1962) is a Ca ²⁺ sensitive photoprotein, ie, it is modified and / or activated upon interaction with Ca ²⁺ . Said modification and / or activation can be detected in particular by non-invasive methods. More specifically, when aequorin interacts with Ca ²⁺ , it binds with two or three calcium ions and then emits a momentary blue light with a maximum wavelength spectrum of 470 nm. In contrast to the classical luciferase-luciferin reaction, no extraneous oxygen is required for light emission, and the total amount of light is related to the amount of protein and the Ca ²⁺ concentration. Oxygen is bound in a molecular form, and aequorin reconstitution occurs by the action of apoaequorin and coelenterazine, a protein with a molecular weight of 21 kDa. Photon emission is caused by peroxidation of coelenterazine after binding to calcium ions on the aequorin protein. Two hypotheses have been suggested for this process: (i) The binding of aequorin and calcium ions causes a conformational change in the protein, which allows oxygen to react with coelenterazine, triggering light emission. (Ii) Oxygen plays a role in the binding of coelenterazine and apoaequorin (Shimomura and Johnson, 1978). Aequorin may be recreated in vitro and in vivo by coelenterazine, for example, by adding it directly to the medium, or by administration, particularly by organ injection (Shimomura and Johnson, 1978).

By replacing the coelenterazine moiety in aequorin with a different coelenterazine analog, up to 30 different semi-synthetic aequorins can be produced (Shimomura, 1995). Different semi-synthetic aequorins show differences in spectra, different Ca ²⁺ binding affinity, stability, membrane permeability and relative regeneration rate. The measurement of Ca ²⁺ concentration can be performed from 100 nM to 1 mM using various combinations. Reconstitution of natural aequorin with natural coelenterazine has a low affinity for Ca ²⁺ (K _d = 10 μM), which makes it a good sensor in the physiological Ca ²⁺ concentration variation range. Although the relationship between light emission and calcium ion concentration may not be linear, the log relationship between light emission and calcium ion concentration has nevertheless been determined (Johnson and Shimomura, 1978). The aequorin consumption rate fraction is proportional to [Ca ²⁺ ] in the physiological pCa range. In fact, when the Ca ²⁺ concentration transitions from 10 ⁻⁷ M to 10 ⁻⁶ M and from 1000 times 10 ⁻⁶ M to 10 ⁻⁵ M, there is a 200-fold increase in signal relative to the background noise ratio. Measured (Cobbold and Rink, 1987). Furthermore, the rate of signal generation is rapid enough to detect an instantaneous increase in Ca ²⁺ ion concentration. An increase in light intensity has been shown with a time constant of 6 milliseconds under calcium saturation conditions (Blinks et al., 1978). Thus, aequorin is a photoprotein that is well suited to measure a rapid and elevated increase in Ca ²⁺ ions under physiological conditions. A recent study examined the CRET response time of a GFP-aequorin reporter containing a linker (Gorokhovatsky et al., 2004). The GFP-Aequorin bioluminescence reaction caused by Ca ²⁺ has been shown to show a typical instantaneous aequorin bioluminescence reaction. After adding Ca ²⁺ to the apparatus where the flow was stopped, light emission started immediately and peaked within 50 milliseconds. The response rate seems to be equivalent to the association rate constant indicated by “chameleon” (Miyawaki et al., 1997).

  Cloning of the apoaequorin gene by Prasher et al. (1985) and Inouye et al. (1985) allows targeting to specific cell compartments by fusion with nuclear, cytoplasmic, mitochondrial, endoplasmic reticulum, or plasma membrane signal peptides Expression vectors have been created (Kendall et al., 1992; Di Giorgio et al., 1996). Furthermore, the in vivo expression of the protein allows its detection at a low level without disturbing the intracellular physiological function of calcium.

Naturally, photoprotein activity is very often associated with a second protein. The most common is “green fluorescent protein” or GFP. In this case, the emitted light is in fact green. The hypothesis of energy transfer between aequorin and GFP by the radiation mechanism was proposed by Johnson et al. (1962) in the 1960s. The blue light emitted by aequorin in the presence of Ca ²⁺ is probably absorbed by GFP and re-emitted in a spectrum with a maximum wavelength of 509 nm. Other studies have shown that this energy transfer occurs by a non-radiative mechanism enabled by the formation of a heterotetramer between GFP and aequorin. Morise et al. (1974) succeeded in visualizing this energy transfer in vitro by simultaneously adsorbing two molecules on a DEAE cellulose membrane. However, these studies showed that the quantum yield of Ca ²⁺ -induced aequorin emission in this condition was 0.23, consistent with that of aequorin alone (Morise et al., 1974).

  GFP has also been isolated in the jellyfish Aequoria victoria, but this has been cloned (Prasher et al., 1992). It has been used in various biological systems as cell expression and lineage markers (Cubitt et al., 1995). Detection of this protein using classic fluorescence microscopy is relatively easy to perform in both living organisms and fixed tissues. Furthermore, fluorescence emission does not require the addition of cofactors or coenzymes and relies on autocatalytic post-translational processes. A 9 amino acid fluorophore forms a ring between serine 65 and glycine 67, which gives the intermediate imidazolidine 5 which is then converted to dehydrotyrosine by oxidation of tyrosine 66 (Heim et al., 1994). This group is found in a cylinder containing 11 β-layers that constitute an environment that interacts directly with the chromophore (Yang et al., 1996).

Real-time monitoring of calcium influx helps elucidate the development, plasticity and function of many organs such as the central nervous system, heart, brain and liver and their associated pathologies. In jellyfish, aequorin protein that chemiluminescents and binds to calcium associates with green fluorescent protein (GFP) and generates a green bioluminescent signal upon Ca ²⁺ stimulation. Aequorin alone is difficult to detect at the cellular and subcellular levels due to weak photon emission after excitation, and extremely difficult to detect at single cell or good temporal resolution.

  A novel marker sensitive to calcium that would have a higher quantum yield is described in WO 01/92300. This marker utilizes chemiluminescence resonance energy transfer (CRET) between two molecules. The calcium sensitive bioluminescent reporter gene is made by fusing GFP and aequorin, which emits much brighter light. Various structures obtained by recombination of nucleic acid molecules encoding GFP linked to aequorin are disclosed in International Patent Application No. WO 01/92300, which is incorporated herein by reference.

The chemiluminescent and fluorescent activities of these fusion proteins in mammalian cells were evaluated. The cytosolic Ca ²⁺ increase was imaged at the single cell level with a cooled sensitized CCD (coupled charge device) camera. This bifunctional reporter gene allows investigation of calcium activity in the neuronal network and specific subcellular compartments of transgenic animals.

The present invention utilizes a fusion protein or recombinant protein made with aequorin and GFP to increase the quantum yield of Ca ²⁺ -induced bioluminescence. This activity cannot simply be increased by co-expressing aequorin and GFP.

Aequorin has a low calcium binding affinity (Kd = 10 μM) and therefore should not have a major effect as a [Ca ²⁺ ] _i buffer system and should not plate the Ca ²⁺ gradient. is there. The rate of signal generation is rapid enough to detect an instantaneous increase in Ca ²⁺ ion concentration with a time constant of 6 milliseconds under calcium saturation conditions. The total amount of light is proportional to the amount of protein and the Ca ²⁺ concentration. It is therefore possible to calibrate the amount of light emitted at any given time to the calcium concentration. Studies have shown that aequorin alone is extremely difficult to detect at the single cell and subcellular levels due to weak photon emission levels.

Binding of Ca ²⁺ to aequorin (having the three EF hand structures characteristic of the Ca ²⁺ binding site) induces a conformational change that oxidizes coelenterazine through an intramolecular reaction. The generated coelenteramide is in an excited state and emits blue light (maximum: 470 nm) when returning to the ground state (Shimomura & Johnson, 1978). When GFP is fused to aequorin by a flexible linker (WO01 / 92300), the energy gained by aequorin after Ca ²⁺ binding is transferred from activated oxyluciferin to GFP without emitting blue light. . The GFP acceptor fluorophore is excited by oxycoelenterazine by energy transfer without radiation. As a result, when the excited GFP returns to its ground state, green-shifted light (maximum, 509 nm) is emitted.

The GFP-aequorin of the present invention is a bifunctional reporter protein that combines Ca ²⁺ sensitivity characteristics and fluorescence of aequorin and GFP. The recombinant protein can be detected by classical epifluorescence in live or fixed samples and can be used to monitor Ca ²⁺ activity by detecting bioluminescence in live samples. The GFP-Aequorin polypeptide is encoded by a gene, and the coding sequence and / or the expressed polypeptide can be localized to a particular cellular domain. The GFP-Aequorin polypeptide can additionally or alternatively be introduced into the organism by gene transfer without disturbing the function of the photoprotein. This nucleic acid encoding the recombinant polypeptide of the invention can also be expressed under the control of a suitable transcription and / or translation system. As used herein, “appropriate” refers to a recombinant polypeptide of the invention in a given cell type, given tissue, given cell compartment (eg, mitochondria, chloroplast, etc.) or given cell domain. It means an element necessary for transcription and / or translation of a nucleic acid encoding a peptide. To achieve the specific expression, the nucleic acid is recombined, for example, under the control of a cell type specific promoter or a tissue type specific promoter, whereby a single cell type or single in a given tissue or animal whole body. Measurement of Ca ²⁺ signaling in one tissue type is possible.

The chemiluminescent and fluorescent activities of GFP-Aequorin protein in mammalian cells were evaluated. The cytosolic Ca ²⁺ increase has previously been imaged at the single cell level using a cooled sensitized CCD (coupled charge device) camera (WO 01/92300). Our study of GFP-Aequorin at the single cell level demonstrated the sensitivity of this recombinant polypeptide for use as a probe (see results below). GFP- aequorin, due to low affinity for ^{Ca 2+,} it does not significantly interfere with the local ^{Ca 2+} signaling. GFP-Aequorin is therefore a bioluminescent reporter of intracellular Ca ²⁺ activity and can be used to track dynamic changes in single cells, tissue sections, or live animals. GFP fluorescence is also a valuable gene expression reporter and intracellular localization marker. Furthermore, since bioluminescent molecules use chemical energy to generate light, they do not require incident radiant energy. Thus, there is virtually no signal background.

  In contrast, fluorescent dyes cannot be localized exclusively in the subcellular domain. On the other hand, chameleons can be targeted genetically. These reporters generally have a low signal-to-noise ratio and long-term imaging is difficult due to problems associated with phototoxicity and photobleaching. This limits the use of these probes, for example in learning and memory, development and cardiac rhythm studies, to visualize long-term dynamic changes. Fluorescence requires radiant energy, which generates photobleaching, phototoxicity, autofluorescence, and a high background signal. An external light source is required to excite the fluorescent molecules. When the excitation light passes through the tissue, it is absorbed and excites the fluorescent molecule. Similarly, the same will occur if the emitted light is detected through tissue. For the time being, non-invasive whole animal imaging in vivo is mainly limited to bioluminescent reporters.

Other related technologies:
Electrophysiological recording is limited to the cell body or large dendritic area that the micropipette approaches.

Yuste et al. Describe the use of fluorescent indicators to detect activation of “subsequent” neurons associated with optical detection of connections between two neurons or between neurons (Yuste et al., 2003). However, this approach has the disadvantage that these fluorescent indicators are only useful for short-term or time-lapse imaging applications since they cannot be gene targeted. In particular, (1) non-selective staining and dye leakage problems from cells after a short time at physiological temperature, (2) live (or fixed) by requiring photoexcitation Long-term dynamic imaging is limited by photobleaching and photodynamic injury caused by dissociated cell cultures or tissue samples. (3) Local Ca ²⁺ dynamics or high-concentration Ca ²⁺ microdomain images by fluorescent dye Is difficult and falls below the limit of spatial resolution provided by optical microscopy techniques.

Voltage sensitive dyes are considered as a useful technique for monitoring “multi-neuronal activity in the intact central nervous system” (Wu et al., 1998). These probes are fluorescent and are therefore subject to the same limitations as discussed for Ca ²⁺ sensitive fluorescent dyes (see Knofpel et al., 2003 for review). Genetically codeable forms have also been developed (Siegel & Isacoff, 1997), but the signal to noise ratio is low.

“Chameleon” belongs to a genetically encoded Ca ²⁺ sensitive fluorescent probe consisting of two GFPs covalently linked by a calmodulin binding sequence. Chameleons generally have a low signal to noise ratio and long term imaging is difficult due to problems associated with phototoxicity and photobleaching. Targeting this probe is a fusion with a transmembrane protein known as fogrin on the surface of a large, dense secretory vesicle in the mitochondrial matrix (Fillipin et al., 2003), in the endoplasmic reticulum cavity (Varadi & Rutter, 2002). (Emmanouilidou et al., 1999).

  The present invention detects single-cell types (neurons and neuronal networks) and calcium dynamics in other organs and tissues in real time to detect calcium signaling microdomains associated with synaptic transmission (single cell types ( A novel approach using combined fluorescence / bioluminescence imaging of neurons and neuronal populations) is described.

In particular, the present invention provides recombinant polypeptides useful for the detection of Ca ²⁺ microdomains. The recombinant polypeptide includes a bioluminescent polypeptide optionally fused to a peptide or protein that can be targeted to the subcellular domain. In one embodiment of the invention, the bioluminescent polypeptide includes chemiluminescent and fluorescent peptides that bind to calcium ions. In another example, the recombinant polypeptide consists of a chemiluminescent peptide that binds calcium ions and the fluorescent peptide. The recombinant polypeptide also consists of a chemiluminescent peptide, a fluorescent peptide, and a linker, optionally further fused to a peptide or protein that can be targeted to the subcellular domain. In certain embodiments, the recombinant polypeptide consists of a chemiluminescent peptide, a fluorescent peptide, and a peptide or protein that can be targeted to the subcellular domain. Another example consists of a chemiluminescent peptide, a fluorescent peptide, a linker, and a peptide or protein that can be targeted to the subcellular domain.

  Targeting of GFP-Aequorin (GA) to subcellular compartments or cellular microdomains is made possible by fusing with a peptide signal or peptide or protein of interest.

  According to certain embodiments, the present invention targets bifunctional fluorescent / bioluminescent recombinant protein (GFP-Aequorin) to detect calcium signaling in cellular compartments, particularly calcium microdomains associated with synaptic transmission Describe the conversion and use. The invention also describes the use of these recombinant polypeptides for “real-time” optical detection of calcium dynamics in single cells or cell populations, such as single neurons and neuronal populations. These studies describe the use of this recombinant polypeptide in neurons, but are also to highlight the sensitivity and other important features provided by this reporter polypeptide. The use of this recombinant polypeptide is certainly not limited to use in neurons. GFP-Aequorin has great utility in other cell types, but certainly in most animals, plants, bacteria and also yeast, especially in any biological system where calcium signaling is important.

  An example of a chemiluminescent peptide is aequorin or an aequorin variant. In a preferred embodiment, the mutant aequorin has a different, preferably low affinity for calcium ions, such as mutant aequorin Asp407 → Ala. In another example, h-coelenterazine analogs may be used to regenerate aequorin protein, which may show higher affinity.

  Examples of fluorescent peptides are green fluorescent protein (GFP), GFP variants, or GFP variants. Such variants have the characteristic of emitting photons at different wavelengths. Examples of such GFP variants are CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), and RFP (red fluorescent protein).

  A variant or variant of a chemiluminescent or fluorescent peptide is defined herein as a sequence having a substitution, deletion, or addition with reference to a reference sequence. Amino acid substitutions may be conservative, semi-conservative, or non-conservative.

  Thus, a particular recombinant polypeptide consists of aequorin and GFP, in particular the fusion polypeptide GFP-Aequorin, with or without a linker. Within the scope of the present invention, a cyan fluorescent protein and aequorin fusion (CFP-Aequorin), a yellow fluorescent protein and aequorin fusion (YFP-Aequorin), a red fluorescent protein and aequorin fusion (RFP), or these Triple fusions comprising any combination of (eg, RFP-YFP-Aequorin), as well as the red-shifted form of GFP-Aequorin, or the lightness, stability, and / or maturity of the reporter protein Included are variants or variants of the original GFP-Aequorin that have mutations that improve the degree.

The invention also provides a recombinant polypeptide consisting of aequorin, GFP, and a linker, particularly a peptide linker. In a particular recombinant polypeptide, the chemiluminescent peptide is aequorin, the fluorescent peptide is GFP, and aequorin and GFP are linked by a peptide linker that allows chemiluminescence resonance energy transfer (CRET). Peptide linkers enabling CRET preferably comprise or consist of 4 to 63 amino acids, in particular 14 to 50 amino acids. In certain embodiments, such a peptide sequence comprises or consists of the sequence [Gly-Gly-Ser-Gly-Ser-Gly-Gly-Gln-Ser] _n , where n is 1-5 Preferably n is 1 or n is 5.

  In another specific embodiment of the recombinant polypeptide of the invention, the peptide or protein that can be targeted to the subcellular domain is synaptotagmin, PSD95, cytochrome c oxidase subunit VIII, and an immunoglobulin heavy chain or its It is selected from fragments, such as N-terminal fragments.

  The present invention also provides a recombinant polynucleotide encoding a polypeptide of the present invention.

  Furthermore, the present invention also provides a vector comprising the polynucleotide of the present invention and a host cell comprising the recombinant polynucleotide or the vector. The cell may be, for example, a eukaryotic cell or a prokaryotic cell, such as an animal cell, a plant cell, a bacterium, or a yeast.

The present invention relates to a method for optical detection of Ca ²⁺ kinetics in a biological system, said method comprising or consisting of a chemiluminescent protein fused or linked to a fluorescent protein present in said biological system. Monitoring the photons emitted by the Ca ²⁺ sensitive polypeptide. Any polypeptide described in this application can be used to perform the detection method. This method is useful for optical detection of Ca ²⁺ signal propagation to detect intracellular Ca ²⁺ signaling or intercellular communication.

  The method can be used to monitor photon emission in various biological systems, in cells or groups of cells in vitro, in animals or plants expressing the recombinant polypeptide of the invention in vivo, or in vitro. It can be performed in a tissue or cell group of a transgenic animal or plant:

  The method includes administering the recombinant polypeptide or a polynucleotide encoding the recombinant polypeptide to a biological system prior to monitoring of photon emission. In the complete animal system, the recombinant polypeptide or nucleic acid encoding it (or the corresponding vector) is preferably administered by intravenous, intraperitoneal, or intramuscular injection. Administration of the nucleic acid encoding the recombinant polypeptide of the invention can be performed by any suitable means, in particular a recombinant vector, in particular a recombinant viral vector. In certain embodiments of the invention, transgenic non-human animals or plants are provided that are specifically transformed with a nucleic acid encoding a recombinant polypeptide. Transformation may be transient or deterministic. In this case, the recombinant polypeptide of the present invention is expressed from a modified genome of a plant or animal.

  The expression and / or localization of the recombinant polypeptide, when expressed from the genome of a transgenic plant or animal, is a specific tissue, single cell type (eg, neuronal cell, heart cell, or liver cell) or cell compartment or It can be limited to a domain (eg mitochondrion or chloroplast).

  The method can also include administration of a molecule that enables bioluminescence and / or activation of a fluorescent protein prior to monitoring of photon emission. In the GFP-Aequorin reporter protein, the method involves administration of coelenterazine to the biological system in conditions and concentrations that allow activation of the aequorin. The aequorin / coelenterazine system has been disclosed in the art (Shimomura, 1991).

In certain embodiments, the present invention provides methods for detecting or quantifying Ca ²⁺ at the subcellular level. The method comprises expressing the recombinant polypeptide of the present invention encoded by the polynucleotide of the present invention in vivo, in a host cell, particularly in a non-human animal, and visualizing the presence of Ca ²⁺ . In some cases, Ca ²⁺ may be semi-quantified. In a preferred embodiment, the detection is so-called “real time” detection.

The invention also characterizes the developmental form or function of a cell group, tissue, cell, or cell compartment or domain by GFP fluorescence detection, and the cell group, tissue, cell or the cell by the optical detection method of the invention. Methods for identifying physiological and / or pathological processes are provided, optionally including characterization of Ca ²⁺ kinetics in a cellular compartment or domain.

The present invention further relates to a method for identifying physiological and / or pathological processes that may include variations in calcium influx or signaling that deviate from a known normal range, said method comprising Ca ²⁺ kinetics according to the application of the present invention. Including optical detection. Alternatively, the optical detection of Ca ²⁺ kinetics can rather be included as part of the protocol for the identification of such processes, in particular for performing diagnostics or monitoring, for example for monitoring therapeutic responses. .

  Furthermore, the present invention provides a transgenic non-human animal or plant comprising the host cell of the present invention.

  The present invention also provides a transgenic non-human animal that can be used in the above-described optical detection method expressing the above-described genetically encoded recombinant polypeptide of the present invention. In certain embodiments, the recombinant polypeptide is encoded by a polynucleotide, optionally under the control of appropriate transcriptional and translational systems inserted into the genome of the transgenic animal. The non-human animal may be a vertebrate, especially a mammal, such as a primate or rodent. In certain embodiments, the non-human animal is a rat, rabbit, or mouse.

The present invention also relates to a method for producing the transgenic non-human animal of the present invention,
Introducing a DNA construct into a non-human animal embryonic stem cell, where the DNA construct contains a sequence encoding a recombinant polypeptide sensitive to calcium concentration, a so-called transgene? The recombinant polypeptide comprises or consists of a chemiluminescent protein linked to a fluorescent protein, the transgene being placed under the control of a promoter and optionally a conditional expression sequence,
-Selecting a positive clone in which the DNA construct is inserted in the genome of the embryonic stem cell;
Injecting the positive clone into a blastocyst and recovering the chimeric blastocyst, and growing the chimeric blastocyst to obtain a non-human transgenic animal.

In certain embodiments in which expression of the recombinant polypeptide is conditional, the following methods of producing transgenic non-human animals can be used:
Introducing the DNA construct into a non-human animal embryonic stem cell, wherein the DNA construct comprises or consists of a sequence encoding a recombinant polypeptide sensitive to calcium concentration, a so-called transgene. The recombinant polypeptide comprises or consists of a chemiluminescent protein linked to a fluorescent protein, the transgene being placed under the control of a promoter and optionally a conditional expression sequence;
-Selecting a positive clone in which the DNA construct has been inserted into the genome of the embryonic stem cell;
Injecting the positive clone into a blastocyst and collecting the chimeric blastocyst,
-Breeding the chimeric blastocyst to obtain a non-human transgenic animal;
Crossing the resulting first transgenic non-human animal with an animal expressing an endonuclease acting on a conditional expression sequence in a tissue or cell in which the expression of the recombinant polypeptide is required. ,
-Recovering transgenic non-human animals expressing the recombinant polypeptide in specific tissues or cells.

  “Conditional” as used herein means that a recombinant protein sensitive to calcium concentration of the present invention is expressed at selected time points during the entire development period of a non-human transgenic animal. Thus, in certain embodiments, a recombinant protein is expressed when a recombinase catalyzes recombination of a conditional expression sequence, eg, a Cre enzyme catalyzes recombination of a Lox recognition site.

  Expression of a recombinase or endonuclease such as Cre can be spatially and temporally regulated by a promoter located upstream of the nucleic acid encoding the recombinase. The promoter may be a cell-specific promoter that allows recombinase expression in, for example, liver cells, heart cells or brain cells.

  In certain embodiments, recombinase expression may be activated by natural or synthetic molecules. Therefore, ligand-dependent chimeric Cre recombinases such as CreERT or CreERT2 recombinases can be used. It consists of Cre fused to a modified hormone binding domain of the estrogen receptor. CreERT recombinase is inactive but can be activated by the synthetic estrogen receptor ligand tamoxifen, thus allowing temporal control from outside of Cre activity. Indeed, by combining tissue specific expression of CreERT recombinase with recombination of a conditional expression site that is its tamoxifen-dependent activity (eg, a Lox site), tamoxifen can be administered to animals both spatially and temporally. Can also control DNA.

  The invention also relates to the progeny of the non-human transgenic animals of the invention. These offspring may be obtained by crossing a transgenic animal of the invention with a mutant disease animal or model.

In a further embodiment of the invention, there is provided a method of screening a molecule of interest to assay the ability to modulate Ca ²⁺ transients, the method comprising:
a) detecting Ca ²⁺ kinetics in a transgenic animal expressing a recombinant polypeptide sensitive to calcium concentration by the optical detection method of the present invention,
b) administering or expressing in the transgenic animal the molecule of interest;
c) repeating step a);
d) the injection position of the front and rear of the ^{Ca 2+,} kinetics, optionally comparing the amount, where the position of ^{Ca 2+,} kinetics, and / or the amount of variation that molecule is a ^{Ca 2+} transients can modulate (the modulate) It suggests,
including.

The present invention also provides a recombinant peptide composition that can be expressed in vivo in a non-human animal by GFP, aequorin, a peptide or protein-encoding polynucleotide capable of targeting the recombinant peptide to the cellular domain. I will provide a. The peptide composition is involved in the visualization or quantification of Ca ²⁺ changes in a cell, group of cells, subcellular domain or tissue of interest.

The present invention provides a means to genetically target the bioluminescent reporter GFP-Aequorin to a variety of microdomains, including microdomains important in synaptic transmission. GFP-Aequorin has an excellent signal-to-noise ratio and can be targeted to proteins or cell compartments without disrupting photoprotein function. Thus, the present invention provides for “local Ca ²⁺ kinetics” at the molecular, cellular, tissue, and whole animal level, or in dissociated cell cultures, dissected tissue, acute and organotypic cultures or live animals. Enables “real time” visualization.

The reporter of the present invention enables selective detection of subcellular or high concentration Ca ²⁺ microdomains. Therefore, the genetically encoded bioluminescent Ca ²⁺ reporter GFP-Aequorin is intended to optically detect synaptic transmission and to facilitate the mapping of functional neural circuits in the mammalian nervous system. Can be used.

Whole animal bioluminescence imaging represents a very important non-invasive strategy for monitoring biological processes in living untreated animals. To date, applications describing the imaging of cellular activity in vivo using bioluminescent reporters have been almost exclusively performed using the firefly luciferin-luciferase system. The approach utilizes a luciferase reporter system for internally generated light linked to a specific biological process. Bioluminescent reactions usually involve the oxidation of an organic substrate (luciferin or chromophore). When cells expressing luciferase combine with the substrate luciferin, light is generated (maximum is 560 nm). Both ATP and O ₂ are required for the photoreaction to occur. Since these reporters were developed to produce red-shifted light at longer wavelengths, the resulting light is less absorbed by the tissue, which allows the technique to track tumor progression or infection Ideal for.

The aequorin-based system offers an alternative to the luciferase-based reporter system for BLI. Light is generated in the presence of Ca ²⁺ and the substrate coelenterazine. In contrast to the luciferin-luciferase system, the Ca ²⁺ -dependent light emission of GFP-Aequorin (maximum 515 nm) does not require exogenous O ₂ . In contrast, molecular O ² is tightly bound, and therefore it is possible for a luminescent reaction to occur in the complete absence of air. Therefore, the bioluminescence reaction rate of the photoprotein is not affected by the oxygen concentration. The second characteristic of aequorin is that the light intensity can be increased to 1 million times or less by adding calcium. Therefore, the coelenterazine-GFP-aequorin system may be more appropriate than the firefly system as a reporter because it allows specific analysis of Ca ²⁺ activity and does not require the cofactors ATP and Mg ²⁺ . Since the red light is emitted by the luciferase reaction, the coelenterazine-GFP-aequorin system is more suitable for deep tissue analysis and is therefore ideal for following the infection process or tumor progression. However, the aequorin-based system can be used to monitor Ca ²⁺ -dependent biological processes at more superficial tissue sites with spatial and fine temporal resolution (mammalian cortex, skeletal muscle or Analysis of Ca ²⁺ signal in skin). With the development of new devices, the GFP-Aequorin-Coelenterazine system can be imaged in the deep tissue layer in the same way as luciferase-luciferin. Therefore, in vivo imaging of the whole animal GFP-Aequorin-Coelenterazine system makes it possible to examine dynamic biological processes in human ecology and live animal models of disease.

  The inventors have developed transgenic mice that express various GFP-aequorin reporter polypeptides. Expression of the nucleic acid encoding this recombinant polypeptide of the invention is driven by appropriate transcriptional and / or translational elements, which are preferentially located upstream of the nucleic acid but located downstream. Also good.

These reporter mice offer a number of advantages over other gene-based or gene-based reporters. This is because it can report multiple activities non-invasively in a living sample and can be realized in vivo. A preferred embodiment is a transgenic mouse that expresses GFP-aequorin targeted to mitochondria, whereby mitochondrial Ca ²⁺ activity can be monitored by bioluminescence imaging, GFP fluorescence can be visualized and reporter expression Determine location and study specific morphological features. Mitochondrial function is a useful biosensor of cellular activity, particularly in the following pathological processes:

  Transgenic animals expressing the GFP-Aequorin reporter can be used to make a diagnosis, to screen for new drugs, or to evaluate a therapeutic response in preclinical studies. Transgenic animals expressing GFP-Aequorin reporter can also be crossed with transgenic disease animal models to study pathological processes. For example, transgenic mice expressing mitochondrial targeted GFP-aequorin in all cells or selected cell types can be crossed with a transgenic Alzheimer's disease mouse model to assess pathological processes or to make new diagnoses. Can be performed or evaluated for treatment response in preclinical studies. Excised tissue and / or dissociated cells obtained from transgenic animals expressing the GFP-Aequorin reporter can also be used to discover new drugs, to assess the activity of existing drugs, or to preclinical studies It can be applied to high-throughput screening assays to assess therapeutic response in or to make a diagnosis.

  A problem to be solved in the production of transgenic animals is the expression of the inserted transgene, which must be sufficiently efficient in producing the encoded protein. This can be done by inserting a polynucleotide encoding a recombinant protein sensitive to calcium concentration or the corresponding DNA construct into the transcriptionally active region of the genome of the animal to be transformed, or by a correct gene such as a reconstituted HPRT locus. This may be done by reconfiguring a particularly favorable environment in which expression is ensured. Insertion relies on any technique known to the person skilled in the art, in particular by homologous recombination.

  Another problem faced is the specific expression of this recombinant protein in tissues, organs or cell types. This can be achieved by using a recombinant system that includes a conditional expression sequence and a corresponding recombinase (eg, endonuclease). A specific conditional expression sequence is a Lox site and the corresponding endonuclease is Cre, which is preferentially expressed under the control of a cell or tissue specific promoter.

In vivo imaging of GFP-Aequorin according to the present invention has been shown to be non-invasive, physiological processes governing function in living animals, pharmacokinetics, pathological aspects of biomolecular processes and others Can be used for monitoring aspects of The detection of Ca ²⁺ can be used to evaluate and monitor many different cell signaling pathways when studying various pathologies, drug effects, and physiological processes. The detection of Ca ²⁺ is a useful diagnosis in many pathological conditions, including cancer, infectious processes, neurological diseases, muscular diseases (eg muscular dystrophy) and for the treatment and diagnosis of cardiovascular diseases. The GFP-Aequorin technology is also applicable to all mammals and other animals such as worms (eg nematodes), fish (eg zebrafish), frogs (Xenopus spp.) And flies (Drosophila spp.).

For example, calcium imaging provides an alternative technique for monitoring liver, heart, or brain activity. For example, Ca ²⁺ signals are linked to neuronal electrical activity and activity via signaling between glia cells, glia cells to neurons, between neurons, or neurons to glia cells (eg, chemical and gap junction junctions) Linked to the propagation of In addition, Ca ²⁺ is involved in many intracellular signaling pathways. The spatiotemporal profile of Ca ²⁺ in the cellular microdomain regulates the activation of key signaling pathways. Thus, by genetically localizing reporters in nanodomains or microdomains, cellular events can be detected in real time even when there is little spatial resolution, such as in whole animal imaging. Monitor at the molecular level. Described below are multifunctional reporter mice for in vivo, in vitro, and in vitro imaging in research and development applications.

Materials and Methods Generation of targeting vectors (Figures 1 and 2)
GA represents an untargeted GFP-Aequorin, referred to as G5A, which contains a 5 repeat flexible linker between the two proteins. The generation of GFP-Aequorin (GA) and synaptotagmin-G5A (SynGA) has been previously described (WO01 / 92300). In order to target the GFP-Aequorin chimera to the postsynaptic domain, we created a fusion of the N-terminal region of PSD95 and GA. In this construct, the entire PSD95 gene (FIG. 12) was cloned into HindIII / EcoRI of pGA (CNCM I-1507, deposited on June 22, 2000) to obtain plasmid PSDGA. It was. A flexible linker consisting of the following sequence 5′A ATT CGG TCC GGC GGG AGC GGA TCC GGC GGC CAG TCC CCG C′3 (FIG. 11) was then added between PSD95 and the start site of GFP.

  By cloning the reporter gene into the PstI / XhoI site of a vector (pShooter, manufactured by Invitrogen) containing a targeting sequence capable of excising the subunit VIII of cytochrome c oxidase, a plasmid mtGA was obtained, and a GFP-aequorin chimera (GA) Was targeted to the mitochondrial matrix (FIG. 12). In addition, GA was targeted to the ER cavity by cloning in frame to the N-terminal region of an immunoglobulin (IgG) heavy chain gene consisting of the VDJ and CH1 domains of the leader sequence. The coding sequence for the N-terminal region of the IgG heavy chain gene was removed from plasmid erAEQmut (contributed by Dr. J Alvarez (Universidad de Valladolid, Spain) with NheI and HindIII). The gene insert fragment was ligated in frame to the N-terminus of the G5A gene in the pEGFP-C1 vector (Clontech) to obtain plasmid erGA.

Mutations that reduce the Ca ²⁺ binding affinity of the photoprotein (Asp407 → Ala) (Kendall et al., 1992) were made in each targeted GA construct by PCR and the plasmids mtGAmut, erGAmut, SynGAmut, and PSDGAmut. Got. All constructs are under the control of the human cytomegalovirus promoter (pCMV). All sequences have been confirmed by DNA sequencing.

Single-cell bioluminescence studies (Figures 5-9)
The culture solution was placed in a glass bottom dish and fixed on a stage adapter of a fully automated inverted microscope installed in a black box. GFP-Aequorin was reconstituted with 2.5-5 μM coelenterazine at 37 ° C. for 30 minutes. Cells that had been transfected with SynGA and coelenterazine were incubated together at room temperature to reduce photoprotein consumption during the reconstitution process. Cells were perfused with Tyrode's buffer. Cells were perfused for 1 minute in the absence of Mg ²⁺ prior to application of a solution of NMDA. All recordings were performed at room temperature (22-25 ° C.). Cells with a cell phase diameter of 10-15 μm with a bright phase and no granular appearance were selected for measurement. Cells transfected with SynGA and reconstituted with wild-type coelenterazine regularly exhibited low levels of bioluminescence activity in the quiescent state. The activity appeared to be uniform and more apparent in the cell body region. This suggests that the GA targeted in this way is in a domain conferred with a high concentration of Ca ²⁺ . In two cases, cells transfected with PSDGA showed spontaneous activity, which was localized in the dendritic region.

Imaging Ca ²⁺ dynamics in organotypic slice cultures (FIGS. 10 and 19)
Organotype hippocampal slices were prepared from 4-5 day old pups. Briefly, brains were quickly removed in ice-cold Hank buffer and sliced into 200 μm sections with a tissue chopper. Hippocampal slices were identified using a stereomicroscope and transferred to a sterile transwell collagen coated chamber (12 mm diameter, 3.0 μm pore size). Sections were maintained at 37 ° C. in neural basal medium supplemented with B27 in a humidified atmosphere containing 5% CO ₂ . Sections were infected on day 9 with the adenovirus-GFP-aequorin vector and maintained in culture for an additional 4 or 5 days before imaging. At this stage, the sections appeared healthy and individual cells could be clearly distinguished by GFP fluorescence. After incubation with coelenterazine, the sections can be maintained on an inverted microscope for up to 9 hours at room temperature, at which time cell death is revealed, indicated by a large increase in bioluminescent activity and loss of cell fluorescence. It was. Since no photoexcitation is required to detect the Ca ²⁺ reporter, long-term imaging can be performed without causing photodynamic injury. Long-term imaging is continuous. There is no need to select an integration period. The background is very low in the 256 × 256 pixel area (665.6 × 665.6 μm) and less than 1 photon / second. In some experiments, 400-450 μm coronal sections of somatosensory cortex of transgenic mice were excised using a vibrator and placed in culture for 4-5 days prior to imaging. Sections were perfused with ACSF bubbled with 95% O ₂ and 5% CO ₂ . After 1 hour incubation, the sections were perfused with ACSF without Mg ²⁺ . Recorded at room temperature (25-28 ° C). Drug was added through the perfusate.

Production of transgenic animals (FIGS. 15-23)
We have a new genetically engineered reporter molecule, according to which GA is targeted to the subcellular domain of transgenic mice. The GA transgene can be expressed in any cell type and / or at any stage of development. Expression was conditioned using the Lox-stop-Lox sequence immediately following the strong promoter β-actin (CAG). Selection of expression cells is done using an appropriate endonuclease Cre driven by a specific promoter. The point at which transcription is initiated is initiated by injecting tamoxifen when using the gene Cre-ER ^T2 . Finally, the transcription unit was introduced by homologous recombination into the reconstituted HPRT locus (X chromosome) of ES cells so that it could be expressed in any cell, minimizing the effect of the integration site on the expression level. In the experiments presented here, PGK Cre transgenic mice that activate the GA transgene in very early embryos were used.

  Recombinant viruses containing CRE under the control of a cell specific promoter can also be used. Mice were generated by injecting genetically modified ES cells into blastocysts.

Immunolocalization study using mtGA (Figure 18)
Cortical neurons or brain sections expressing GFP-aequorin reporter targeted to mitochondria were fixed in 4% formaldehyde in PBS for 20 minutes at room temperature. After washing with PBS, the cell membrane was permeable with PBS containing 0.1% Triton-X100 and BSA. The cells were then incubated with the primary antibody for 1-2 hours at room temperature. Targeting was compared to anti-cytochrome c (1: 500; BD Biosciences Pharmingen, CA, USA) and Mito Tracker® Red CMXRos (200 nM; Molecular Probes). Antibody binding was measured after incubation for 1 hour in a secondary antibody conjugated to Alexa Fluor® 546 (Molecular Probes). After washing, the cells were placed on a Fluoromount slide and visualized by confocal analysis. Images were acquired with an Axiovert 200M laser scanning confocal microscope (Zeiss LSM-510; version 3.2) through an oil immersion objective (63x / 1.4NA) using LP560 and BP505-550 filters. The pinhole opening was set at 98 Tm and the image was digitized into a 512 × 512 array with 8 bit resolution.

Combined fluorescence / bioluminescence imaging (Figures 5-7, 8-10, 13, 14, and 19)
The fluorescence / bioluminescence wide-field microscope system was manufactured as a special order from ScienecWares. The system contains a fully automated inverted microscope (200M, Zeiss, Germany) and is placed in a dark box that does not transmit light. A mechanical shutter controls illumination from both halogen and HBO arc lamps installed outside the box and connected to the microscope via fiber optic cables. Low level light emission (photon velocity <100 kHz) is collected using an Image Photon Detector (IPD3, Photek) connected to the base port of the microscope, which contains the X, Y coordinates of each detected photon and Assign time points (Miller et al., 1994). The system is fully controlled by data acquisition software, which also converts a single photon event into an image that is generated by a bright field or fluorescent image created by a CCD camera connected to the C port. (Coolsnap HQ, Roper Scientific). Therefore, any Ca ²⁺ activity that can be visualized can be analyzed in more detail by selecting a region of interest and outputting photon data. After the experiment is complete, you can play the recorded movie file and extract the data according to your needs. The IPD can provide sub-millisecond time resolution and the integration time need not be explicit for acquisition. The system we use has a very low background photon count level of <1 photon / second in a 256 × 256 pixel area.

Calibration of bioluminescence measurements to intracellular Ca ²⁺ values of living cells can be performed by in vitro calibration. Intracellular [Ca ²⁺ ] measurements were made by determining the photoprotein consumption rate fraction. Neuro2A cells were transiently transfected with different constructs for in vitro calibration. After 48 hours, cells were washed with PBS and harvested using a cell scraper. The cell suspension is transferred to a 1.5 ml Eppendorf tube and 2 ° C. at 4 ° C. in aequorin reconstitution buffer containing 10 mM mercaptomethanol, 5 mM EGTA, and 5 μM wild type (wt) n or h coelenterazine in PBS. Incubated for hours. After 2 hours, the cells were washed and a hypotonic buffer containing 20 mM Tris / HCl, 10 mM EGTA, and 5 mM mercaptoethanol in dH ₂ O and a protease inhibitor without EDTA (Roche Diagnostics). Resuspended in. The cell membrane was lysed by three additional freeze-thaw cycles, after which the suspension was passed through a 26GA needle. A 10 μl aliquot of cell lysate containing reporter protein was dispensed into wells of a white opaque 96 well plate containing EGTA buffer solution with known concentrations of CaCl ₂ (Molecular Probes). Free Ca ²⁺ was calculated using the WEBMAXC program (www.stanford.edu/~cpatton/webmaxc.html) (Bers et al., 1994). Luminescence was measured directly using a 96 well plate reader (Mithras, Berthold Tech., Germany). Light was recorded for 10 seconds with 100 millisecond integration after cell lysate injection. After 10 seconds, 100 μL of 1M CaCl ₂ solution was injected into the same well and recording continued until light returned to basal levels and all photoprotein was consumed (Lmax). Light emission is expressed as a fraction of the rate of photoprotein consumption, which is the light emission (L, s ⁻¹ ) at that time (defined [Ca ²⁺ ]) and until the photoprotein is completely consumed. Ratio between integrals of total light emission at time (Lmax) (saturated Ca ²⁺ ). The experiment was performed at 25-28 ° C.

Electrical Stimulation and Virus Transfection In some experiments, an electrical pulse was stretched from a borosilicate glass (World Precision Instruments, Florida, USA) through a classic patch pipette (5-10 MΩ) under test. Delivered to cells. An electrically operated relay system created in the laboratory allowed the measurement of pipette resistance between stimuli delivered by isolated stimulators (DS2A, Digitimer, UK) (see FIG. 14). Ca ²⁺ wave propagation velocity was calculated by taking the time point corresponding to half the maximum value of light emitted after stimulation and dividing by the measured distance between the centers of the two regions analyzed.

Whole animal bioluminescence detection Natural coelenterazine (4 μg / g body weight; Interchim, France) was introduced into P1-P4 mice by intraperitoneal injection. Mouse imaging began 1 to 1.5 hours after substrate (coelenterazine) injection. The IVIS® imaging system 100 series, which enables real-time imaging and can monitor and record cellular activity in living organisms, is used in these studies to produce local Ca at the whole animal level. ²⁺ changes were detected. The system features a cooled back-thin, back-illuminated CCD camera in a low-background imaging chamber that does not pass light. A gray scale surface image of the mouse was first acquired using a 10 cm field of view, an exposure time of 0.2 seconds, a binning resolution factor of 2, 16 f / stop (opening) and an open filter. A bioluminescent image was acquired immediately after the grayscale image. The bioluminescence image acquisition time ranged from 1 to 5 seconds, binning 8 and 16, imaging field of 10 cm; f / stop1. The relative intensity of light transmitted from bioluminescence in vivo was displayed as a false color image ranging from purple (lowest intensity) to red (highest intensity). Corresponding grayscale photographs and colored luciferase images were overlaid using LivingImage (Xenogen) and Igor (Wavemetrics, Lake Oswego, Oreg.) Image analysis software.

Results Result 1: Targeting GA to the subcellular domain In order to express in specific subcellular compartments, various GA reporters were made by fusing with the signal peptide or protein of interest (Figures 1 and 2). GA was targeted to domains important for synaptic transmission: mitochondrial matrix, endoplasmic reticulum, synaptic vesicles, and post-synaptic thickening. By confocal analysis, fused to cytochrome c signal peptide for targeting to the mitochondrial matrix (mtGA), then fused to IgG heavy chain for targeting to the ER cavity (erGA), synaptic vesicles Expected GA after fusing to synaptotagmin I protein for targeting to the cytosol side of the membrane (SynGA) or after fusing to PSD-95 for targeting to post-synaptic thickening (PSDGA) Reporter expression patterns were shown (Figure 4) (Christopherson et al., 2003; Conroy et al., 2003).

Results 2: GA reports Ca ²⁺ concentrations by single-cell resolution We started these studies with untargeted GAs that are uniformly distributed in neurons (Fig. 5). After reconstitution of GA with h-coelenterazine (high affinity form of luciferin) and stimulation with NMDA (100 μM) and KCl (90 mM), a strong signal was generated in cortical neurons (FIGS. 5B and C). This was the first time that Ca ²⁺ responses in small mammalian neurons were visualized directly using bioluminescent reporters at the single and subcellular levels. Application of digitonin and a high concentration of Ca ²⁺ at the end of the experiment showed that a sufficient amount of photoprotein remained (FIG. 5D). High concentrations of Ca ²⁺ and digitonin were also added at the end of the experiment to measure the total available GFP-Aequorin (Lmax) to normalize the data (see definition of Lmax in the method section).

  Results 3: Optical detection of GFP-Aequorin targeted to synaptic proteins involved in calcium signaling

High concentrations of Ca ²⁺ microdomains were detected using targeted GFP-Aequorin after stimulation of cortical neurons.
Example 1: When GA was targeted to a compartment such as the mitochondrial matrix, differences in the kinetic properties of the Ca ²⁺ response could be detected under the cells (FIG. 6). A representative example was shown in which application of NMDA caused a Ca ²⁺ response at defined cell locations with different time profiles. FIGS. 6A and B show that the Ca ²⁺ response in a particular region could be analyzed despite low levels of light emission and moderate spatial resolution compared to conventional fluorescence. When the reporter was targeted under the cells, a signal that was 1000 times smaller than the background could still be detected (15 × 15 pixel area, 1 pixel = 0.65 μm). The graph data derived from the local regions shown in the graph demonstrates the diversity of spatiotemporal characteristics of mitochondrial Ca ²⁺ changes.

Example 2: Optical detection of Ca ²⁺ -induced bioluminescence in neurons using GFP-Aequorin targeted to synaptotagmin I, a synaptic vesicle transmembrane protein of the calcium sensor. See Figures 1, 2, 24D and 7. Synaptotagmin I is a low affinity Ca ²⁺ sensor that is thought to be involved in the regulation of rapid exocytosis events (Davis et al., 1999). Previous studies have shown that synaptotagmin I is “tuned” to respond to Ca ²⁺ concentrations (21-74 μM) that cause synaptic vesicle membrane fusion (threshold> 20 μM, maximal at 194 μM). Half speed). We have created a low affinity form of GFP-Aequorin, known as SynGAmut, that is targeted to synaptic vesicles by fusion to synaptotagmin I. It should be possible to optically probe neuroexocytosis specifically by using a low affinity Ca ²⁺ reporter that detects only the high concentration calcium domain. For example, SynGAmut allowed specific detection of vesicles located near voltage-gated Ca ²⁺ channels that dock for neurotransmitter release (FIG. 4). These vesicles are located close enough to the channel mouth and are therefore located in the high concentration Ca ²⁺ domain, which we believe is necessary to drive vesicle exocytosis that facilitates synaptic transmission. It has been. Because the reporter has a low affinity for Ca ^2+, vesicles that are not docked for release will not be detected far enough.

Example 3: Ca ²⁺ -induced bioluminescence can also be visualized by subcellular resolution in cortical neurons expressing PSDGA and mtGA after NMDA application (Figures 7 and 8). In contrast to GA, application of NMDA to PSDGA-transfected neurons resulted in Ca ²⁺ transients with faster response rates and larger amplitudes (see FIG. 8 for a representative example of 3 experiments). . A clear difference in Ca ²⁺ kinetics was observed between dendrites (FIGS. 8D1 and D2) and cell bodies (FIG. 8, cell bodies). In particular, the analyzed dendritic regions showed a faster rate of rise and rapid decay of the Ca ²⁺ response. The rapid decay rate suggested that the photoprotein was completely consumed and that the temporal kinetics and amplification of the Ca ²⁺ response was artificial. Furthermore, no photoproteins were available any longer, suggesting that the Ca ²⁺ concentration in the domains targeted by PSDGA would have been very high (for the Ca ²⁺ binding curve). (See FIG. 3).

Example 4: When cortical neurons were transiently transfected with PSDGA, GA was locally expressed in dendritic structures (Figures 8 and 9). By targeting GA locally when fused to PSD-95, a special type of activity could be observed in cortical neurons that remained in the basal state. We, in contrast to SynGA, where the rate of light emission is constant, Ca ²⁺ transients that are higher than the very low background that was spatially localized in some experiments. Was observed. In at least two experiments (representative example shown in FIG. 9), calcium transients occurred relatively frequently and were confined to the dendritic region (see FIGS. 9A, C, and B).

Example 5: Intracellular Ca2 ⁺ propagation in PSDGA-transfected cortical neurons (Figure 13)

  Results 4: Use of genetically targeted GFP-Aequorin for “real time” visualization of calcium dynamics in neuronal populations to map neural connectivity

We have verified the use of these reporters to track cell-to-cell transmission in cultured neurons. We also electrically stimulated hippocampal neurons expressing GA reporters to visualize the propagation of Ca ²⁺ activity and cellular transmission within simple neural networks. In these experiments, we used a replication-defective adenoviral vector (Ad5-GA) encoding GA to transfect dissociated hippocampal neurons. FIG. 14F1 shows at least two cells expressing the GA reporter. When a short electrical pulse was applied to the body region of a cell labeled with I (FIG. 14BF), a Ca ²⁺ wave propagated to the cell labeled with the next III. Analysis of the Ca ²⁺ response in three regions suggested that Ca ²⁺ propagation fluctuates between each region. In the region from I to II, the waves were calculated to travel at a speed of 60 μm / sec. In contrast, in the region from II to III, it was calculated to travel at a speed of 10 μm / sec. A total of 6 consecutive stimuli (one single pulse about every 2 seconds) was applied (of which the first 5 were graphed), after which spontaneous oscillations appeared (Figure 14A-F). Each Ca ²⁺ “spike” showed a rapid rate of rise followed by a slow decline. Even after recording these Ca ²⁺ transients for about 45 minutes, significant photoprotein activity remained (determined by mechanical cell membrane disruption).

Spontaneous activity recorded in organotypic sections infected with a replication-defective adenoviral vector (Ad5-GA) encoding GA (FIG. 10). Long-term recording (up to 8 hours) could be performed when GA was expressed in tissue sections, such as cortical organotypic sections. In general, we find that the sensitivity level for detecting photoprotein consumption and Ca ²⁺ variation is related to the amount of reporter expressed, the location of the reporter, and the type of coelenterazine analog used. (Shimomura, 1997; Shimomura et al., 1993). This may vary from cell to cell depending on the application and needs to be optimized in each case as well as fluorescent probes.

  Result 5: Generation of transgenic animals expressing GFP-Aequorin in specific cell types, subcellular compartments or cellular microdomains for calcium dynamics studies in whole animal studies

Transgenic animals expressing the targeted GFP-Aequorin reporter were generated. The GA transgene can be expressed in any cell type and / or at any stage of development. Expression was made conditional by using a Lox-stop-Lox sequence immediately after the strong promoter β-actin (CAG). Selection of expression cells was done by using the appropriate endonuclease Cre driven by a specific promoter. A transcription unit was introduced into the reconstituted HPRT locus of ES cells by homologous recombination so that it could be expressed in any cell, minimizing the effect of integration sites on expression levels. Recombinant viruses containing CRE under the control of a cell specific promoter can also be used. In the example shown here for transgenic animals made with GFP-Aequorin targeted to the mitochondrial matrix, these mice were crossed with PGK-CRE mice, in all cells and from the onset of development. Activation of transgene expression was induced. Fluorescence imaging of whole embryos and newborn mice revealed that the mtGA transgene was expressed in all cell types (FIGS. 15, 17, 18 and 19). Reporter proteins were also well targeted to the mitochondrial matrix (FIGS. 16C2 and 18). Some advantages of GFP-aequorin are that it is not an endogenous protein normally expressed by mammalian cells and has little effect on Ca ²⁺ signaling due to its low binding affinity. Therefore, the inventors did not find any abnormal phenotypic findings in the transgenic system obtained with the GFP-Aequorin reporter.

  Confocal analysis showed the expected expression pattern of the GA reporter after fusion to the cytochrome c signal peptide for targeting to the mitochondrial matrix (mtGA) (FIG. 16C2). GFP fluorescence images of major organs showed strong reporter expression levels in major organs (FIG. 17). Higher expression levels were found in the heart and liver. In the brain, transgene expression was evident in both glial cells and neurons after comparison with antibodies against GFAP and NeuN, respectively (FIG. 19). These results indicated that when transgenic mtGA reporter mice were crossed with PGK-CRE mice, the mtGA transgene was successfully targeted and expressed in all cells of the brain.

Analysis of luminescence activity indicated that the GFP-Aequorin protein is functioning for the detection of Ca ²⁺ . We obtained preliminary data indicating that we can detect mitochondrial Ca ²⁺ oscillations in neocortical organotypic sections using our transgenic mice (FIG. 20). Ca ²⁺ transients occurred synchronously over a large area at a rate of once every 15-45 seconds. Since bioluminescence is used to detect Ca ²⁺ activity, section imaging could be performed in real time up to 8 hours. Our results indicate that the GFP-Aequorin reporter provides an excellent signal to noise ratio for detecting Ca ²⁺ transients in brain sections of transgenic animals.

Bioluminescence was also detected in transgenic mice expressing GFP-Aequorin targeted to mitochondria. In order to image Ca ²⁺ -induced bioluminescence in transgenic mice expressing the GFP-Aequorin reporter, it was necessary to inject coelenterazine (eg, intraperitoneally or via the tail vein). We tested whether local Ca ²⁺ kinetics could be detected in live mice after intraperitoneal injection of coelenterazine into neonates and then imaging them at various temporal resolutions. Transgenic mice were directly compared to non-transgenic mice by imaging a series of consecutive images of frames taken over 5 seconds. Mouse grayscale photographs were first collected to track mouse movement and these images were correlated with overlays of bioluminescent images. We have found that the continuum file shows dynamic bioluminescence emission correlated with mouse movement (FIGS. 20 and 21). The detected bioluminescence was characterized by light with a short instantaneous reaction rate (appearing in one frame) and corresponding to the movement of the mouse. That 2-4 seconds of the frame, in a more prolonged resolution (Figure 22), Ca 2 ⁺ signals were also detected in freely moving and the mitochondrial matrix in mice. The inventors were also able to detect a signal with a good signal-to-noise ratio using a 1 second imaging time (FIG. 23). Short instantaneous bioluminescence was detected in areas such as the hind paws, forefoot, spinal cord, brainstem, and other back areas of the body. In all cases, these Ca ²⁺ signals were in sync with the regions of the body undergoing skeletal muscle contraction-relaxation as seen in the grayscale photographs taken before each luminescent image. Using in vivo fluorescent Ca ²⁺ reporter analysis by two-photon microscopy, skeletal muscle mitochondria have only recently been shown to take up and release Ca ²⁺ during muscle contraction-relaxation. However, this technique was more invasive. Because it uses excitation light and electroporation techniques as a means of delivering DNA to muscle fibers, it dismounts distant tendons for in vivo imaging using a two-photon microscope, This is because it is necessary to surgically expose the muscle fibers. In addition, there is some evidence suggesting that mice need to be kept under anesthesia during surgery, and that volatile anesthetics may directly affect complex proteins in the mitochondrial respiratory chain. is there. Nevertheless, these studies yielded images of mitochondrial Ca ²⁺ uptake and release during muscle fiber contraction-relaxation with very high spatial resolution.

  Potential Application 1: Detection of calcium fluctuations associated with morphological or developmental changes in neurons using genetically targeted GFP-Aequorin

Ca ²⁺ is thought to be a central modulator of growth cone motility in neurons. Use photolabile caged (photolabile caged) Ca ^2+, studies, transient rise in Ca ²⁺ has been shown to modulate the growth cone motility positively. GAP43 is thought to be an important protein involved in developing axon guidance and regeneration after nerve injury. GAP43, an axon / growth cone protein, is thought to be activated by a transient Ca ²⁺ increase that associates with calmodulin at the membrane and is thought to be associated with growth cone motility. Thus, GFP-Aequorin targeted to the postsynaptic thickening protein GAP43 can monitor local calcium signaling and corresponding morphological changes associated with neurite outgrowth during development and nerve regeneration It was.

Potential Application 2: Using Targeted GFP-Aequorin to Monitor Receptor Function, Ca ²⁺ Permeability, or Related to Local Increases in Calcium Concentration

Example 1: Use of GFP-Aequorin fused to PSD95 to specifically monitor abnormalities associated with local Ca2 ⁺ increase after activation of NMDA receptors and calcium signaling in neurological diseases such as Alzheimer's disease

  Example 2: High-throughput screening of pharmacological agents or chemical compounds or combinatorial compound libraries for detection of candidate pharmacological agents capable of treating neurological diseases (ie multiwell format, 96, 386, or 1544 wells) Use of targeted low affinity GFP-Aequorin for the selective detection of high concentration calcium microdomains in cell population studies. This technique is much more powerful than using a fluorescent die that senses calcium.

Possible application 3: Preclinical studies:
Dynamic images of Ca ²⁺ activity can be obtained by bioluminescence imaging of the whole animal of a living subject in vivo (FIGS. 15-18). The resulting light penetrates mammalian tissue and can be detected externally and quantified using a sensitive photoimaging system. The GFP-Aequorin signal generated from within a living animal resulted in a Ca ²⁺ influx image with a high signal-to-noise ratio, which could be tracked with a high temporal resolution. This technique should represent a powerful tool for performing non-invasive functional assays in living subjects and provide more predictive animal data for preclinical study studies.

DISCUSSION Accordingly, the present invention provides a modified bioluminescent system comprising a fluorescent molecule covalently linked to a photoprotein, wherein the linkage between the two proteins stabilizes the modified bioluminescent system. The energy can be transferred by chemiluminescence resonance energy transfer (CRET). In a preferred embodiment, the bioluminescent system comprises a GFP protein covalently linked to an aequorin protein, wherein the linkage between the two proteins has the function of stabilizing the modified bioluminescent system; It allows energy to be transferred by chemiluminescence resonance energy transfer (CRET).

  The present invention provides a composition comprising a recombinant polypeptide, the composition having a functional characteristic that binds calcium ions and makes energy measurable, the energy being non-photoexcitable In the presence, it depends on the amount of calcium bound and the amount of polypeptide in the composition.

  The present invention incorporates a peptide linker that has the function of bringing the donor site closer to the acceptor site under optimal conditions after translation, thereby enabling direct transfer of energy by chemiluminescence in the polypeptide of the present invention. It was. Preferred linkers are described in PCT Application No. WO 01/92300, US Application Nos. 09 / 863,901 and 10 / 307,389, issued Dec. 6, 2001, the entire disclosure of which is incorporated herein by reference. Reliable and incorporated herein by reference.

Thus, the present invention provides the formula:
GFP-linker-AEQ
The recombinant polypeptide is used.

  Where GFP is a green fluorescent protein; AEQ is aequorin; the linker is a polypeptide of 4 to 63 amino acids, preferably 14 to 50 amino acids.

The linker has the following amino acids:
(Gly Gly Ser Gly Ser Gly Gly Gln Ser) _n (where n is 1 to 5)
Can be included. Preferably n is 1 or n is 5. The linker can also include the amino acid sequence Ser Gly Leu Arg Ser.

Another recombinant polypeptide for energy transfer from aequorin to green fluorescent protein by chemiluminescence resonance energy transfer (CRET) after activation of aequorin in the presence of Ca ⁺⁺ is of the formula:
GFP-linker-AEQ
Have

Where GFP is a green fluorescent protein; AEQ is aequorin; the linker is the following amino acid (Gly Gly Ser Gly Ser Gly Gly Gln Ser) _n (where n is 1 to 5)
Wherein the fusion protein has an affinity for Ca ²⁺ ions and a half-life of at least 24 hours. The linker can comprise the amino acid sequence Ser Gly Leu Arg Ser. In addition, the recombinant polypeptide can further comprise a peptide signal sequence for targeting the recombinant polypeptide to a cell or subcellular compartment.

  The present invention also provides a polynucleotide encoding the above-described recombinant polypeptide.

  The plasmid containing the polynucleotide of the present invention is as follows: Collection Nationale de Cultures de Microorganisms (“CNCM”), Pasteur Institute, 28, rue du Docteur Roux, 75724 Paris Cedex 15 Has been deposited in France.

  E. containing PSDGA plasmid E. coli cells can be cultured at 37 ° C. in LB medium under conventional cell culture conditions.

Ca ²⁺ transients are involved in a wide variety of signaling pathways that are required for development, neuroplasticity, neurotransmission, excitotoxicity, and other important processes. Ca ²⁺ -dependent activities encoded at the cellular and subcellular levels are complex and involve spatial, temporal and quantitative factors. Here we have demonstrated a technique for quantifying local Ca ²⁺ signaling by visualizing the bioluminescence of GFP-Aequorin, a genetically encoded recombinant polypeptide. By fusing to signal peptides or proteins important for synaptic transmission, a set of Ca ²⁺ sensitive recombinant polypeptides was created that could be used to directly visualize local Ca ²⁺ signaling in single cells or more complex systems . By detecting bioluminescence, it was possible to measure local Ca ²⁺ signals with different spatiotemporal characteristics with a good signal-to-noise ratio.

  Toxicity and degradation of recombinant GFP-Aequorin polypeptide

  The present invention allows this bifunctional recombinant polypeptide to investigate calcium activity in dissociated cell cultures, acute or organotypic sections and transgenic animals in neural networks, specific subcellular compartments and cellular microdomains. Showed that to do.

Expression of this recombinant polypeptide of the present invention in different biological systems has shown no toxicity in vitro or in vivo, even in single cells, tissues, or even whole transgenic animals or plants. This result was also achieved when the recombinant polypeptide was strongly expressed. Therefore, no toxicity was reported even when the recombinant polypeptide was expressed from the early developmental stage to the adult stage of the transgenic mouse. Furthermore, despite the fact that the recombinant polypeptide is neither an endogenous protein nor an endogenous component, the expression did not disrupt normal physiological function. No behavioral defects were reported. Due to the low Ca ²⁺ binding affinity of GFP-Aequorin (see FIG. 2C), no significant interference with the Ca ²⁺ signal was caused.

  Another feature of this recombinant polypeptide is that although it is of exogenous origin, degradation of the recombinant protein did not occur depending on the cell type or developmental stage being expressed.

The characteristics of these bifunctional recombinant polypeptides have made it a useful tool for studying local Ca ²⁺ signaling during disease processes. In particular, as in the study of NMDA receptor function in a neurodegenerative disease model using PSDGAmut protein targeting to a post-synaptic hypertrophy containing a NMDA receptor using a targeted bifunctional recombinant polypeptide, Receptor function could be monitored when associated with local increases in Ca ²⁺ concentration.

These bifunctional recombinant polypeptides are characterized by neural connectivity with changes in network properties associated with duration of developing neural connectivity or changes associated with learning and memory, aging and chronic drug exposure It becomes a very useful tool for visualization of dynamic or variable changes in Ca ²⁺ at central synapses that can occur over long periods of time, such as

The characteristics of these bifunctional recombinant polypeptides make them extremely useful tools in the diagnostic and drug discovery process. In particular, targeted bifunctional recombinant polypeptides could be used to specifically monitor receptor function associated with local increases in known Ca ²⁺ concentrations. GFP-Aequorin can be targeted to specific cellular sites associated with high concentrations of Ca ²⁺ microdomains and can be used to more specifically monitor drug effects in cell populations using high-throughput screening. It was. Current methods use fluorescent indicators that are not targeted for this purpose and are indirectly related to photobleaching, phototoxicity, low signal-to-noise ratio, die leakage, uneven die distribution, and receptor activation. There are problems associated with detecting other calcium kinetics associated with (eg calcium-induced calcium release). Recent studies of high-throughput drug candidate screening have demonstrated that non-targeted GFP-Aequorin has a significantly better signal-to-noise ratio than that provided by fluorescent probes. This is particularly useful for monitoring low Ca ²⁺ influx, such as Ca ²⁺ influx induced by activation of inhibitory G-proteins involved in many neurological diseases (Niedernberg et al., 2003). Therefore, GFP-Aequorin may be useful for monitoring Ca ²⁺ influx associated with nicotine receptor subtypes, known as α-7-containing nicotine receptors, which are difficult to detect with fluorescent indicators.

Optical detection of Ca ²⁺ can provide a simple technique for visualizing “real-time” dynamic activity and long-term cellular changes associated with a particular phenotype or condition. For example, characterizing the spatial and temporal specificity of the Ca ²⁺ profile in synaptic function is associated with early events associated with the development of schizophrenia and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's chorea. It is important to elucidate the mechanisms responsible for the cellular Ca ²⁺ homeostasis involved (Lidow, 2003; Mattson and Chan, 2003; Stuttzman et al., 2004; Tang et al., 2003).

The advantage of working with bioluminescence is that recordings with very high time resolution can be performed over a long period of time. For long-term recording, it is necessary to consider the consumption of aequorin photoprotein. However, we were able to take long-term recordings (up to 9 hours) with very high temporal resolution (1 millisecond resolution) in tissue sections including cortical sections. Overall, the inventors have determined that the sensitivity level of photoprotein consumption and Ca ²⁺ variation depends on the amount of recombinant polypeptide expressed, the location of the expressed recombinant polypeptide, and the type of coelenterazine analog used. We found it relevant (Shimomura, 1997; Shimomura et al., 1993). This may vary from cell to cell depending on the application and needs to be optimized in each case as well as fluorescent probes. Ca ²⁺ sensitive bioluminescent recombinant polypeptides can also represent reporters selected in the study of light sensitive biological systems.

Comparison with Ca ²⁺ sensitive fluorescent reporter system

  Fluorescence visualization requires an external light source for the excitation of fluorescent molecules by light (light is caused by the absorption of radiation), which is photobleaching, phototoxicity, and autofluorescence (intensities that vary over time) High background). Bioluminescent reporters have a significantly higher signal to noise ratio than fluorescent reporters. In imaging applications, a high signal-to-noise ratio is desirable to obtain better quality data. This is because low level signals are less susceptible to interference.

Bioluminescence detection is a non-invasive method for monitoring biological processes in living untreated animals. Fluorescent reporters are problematic because they produce a large amount of autofluorescence upon photoexcitation on tissue. Furthermore, the light must pass through the tissue to excite the fluorescent molecules, after which the longer wavelength emitted light must return through the tissue and be detected by the detector. Genetically encoded Ca ²⁺ sensitive fluorescent reporters are sensitive to temperature and pH, or contain calmodulin, a natural protein of mammalian cells. Overexpression of calmodulin can produce a phenotype in transgenic animals.

  Compare the aequorin-coelenterazine system to the luciferase-luciferin system for biofluorescence imaging of whole animals in vivo

The luciferase reporter system has been utilized to track infection processes, tumor progression, and gene expression in living animals. This system is now well established as an animal model for testing drug candidates in clinical trials. Our recent data on single cells and brain sections suggests that the use of GFP-Aequorin at the single cell level as a bioluminescent reporter was favorably compared to the luciferase system. Since GFP-Aequorin is a recombinant polypeptide that can be used as a reporter of Ca ²⁺ activity, we have identified mice expressing recombinant polypeptide to track more dynamic changes in live mice. Used and shown to be able to detect GA bioluminescence with good temporal resolution at the whole animal level. Increased [Ca ²⁺ ] _i concentrations in specific cellular domains are associated with the development of many pathological processes. Calcium is also an important signaling ion involved in developmental and apoptotic processes. We found in our studies that large Ca ²⁺ changes are associated with apoptotic events. Early events in this process are thought to be involved in the release of Ca ²⁺ by mitochondria. Since Ca ²⁺ levels in the mitochondria of quiescent healthy cells are generally close to the cytosol [Ca ²⁺ ], an increase in mitochondrial Ca ²⁺ levels appears to indicate a very early change leading to cell death. Mitochondria are now clearly seen as cellular biosensors, and changes in mitochondrial Ca ²⁺ handling are central to this property. This provides an alternative approach to luciferase reporters and broadens the applications that are possible with this technology. Combined with the continuous improvement of detection technology and the improvement of the upcoming cofactor chemistry, mice expressing these recombinant polypeptides have been identified in all aspects of biology for clinical trials of new treatment formats. It has the potential to be a powerful analysis system.

  Finally, we created a transgene that encodes the strong promoter β-actin and also mtGA under the control of the Lox-Stop-Lox system. The results presented here indicated that the CRE-regulated transgene is functional in the mouse embryo. By inducing cell type specific expression of the reporter protein at any stage of development of the mouse, the present inventors have now been able to more accurately study the cellular processes occurring in living animals. For example, we could use a recombinant virus containing a CRE under the control of a cell specific promoter. Therefore, carcinogenic processes or tumor progression could be studied at the selected cell type or tissue, acute or organotypic section, or whole animal level.

  The main advantage is that these animals could be crossed with mutant disease animals or models to investigate different pathologies. These animals can also provide a source of specific labeled tissues, cells and tumors for in vitro or in vitro studies.

Definitions The following terms, as used herein, have the following meanings.

Emission Radiation of UV, visible, or IR electromagnetic radiation from an atom or molecule. This emission is caused by a transition from an electrically excited state to a weaker energy state, generally the ground state.

Fluorescence Fluorescence produced by a very short electrically excited singlet. This emission disappears simultaneously with the excitation source.

Chemiluminescence Luminescence generated by chemical reaction.

Bioluminescence Visible chemiluminescence produced by living organisms. The present invention mimics a naturally occurring system in jellyfish without being fixed to a support.

Bioluminescent system The bioluminescent system according to the present invention is a chimeric trimeric molecule comprising a peptide linker and a coenzyme (ie coelenterazine) in the middle. The first and second molecules covalently attached to the linker can be anything as long as they have first a donor site and second an acceptor site attached to it (receptor-linker- Ligand, antibody-linker antigen). The chimeric proteins are Coen.L., Osta.R., Maury, M., and Brulet, P., Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system., Proc. Natl. Acad. Sci. (US) 94 (1997) 9400-9405 may be fused to a tetanus toxin fragment or a membrane receptor for retrograde synaptic transport via axon.

No radiation When aequorin combines with calcium ions, there is no photon emission from aequorin to GTP. (Therefore, in the present invention, there is no transmission of blue light by aequorin and energy transfer is directly between the two proteins).

FRET system Energy transfer by resonance by fluorescence (ie between two GFP variants).

References Ca ²⁺ fluorescent indicator based on green fluorescent protein and calmodulin.

  Miyawaki, A., LIopis, J, Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M. and Tsien, R.Y., Nature (1997) Vol. 388p. 882-887.

Detection of Ca ²⁺ -dependent changes in fluorescent emission of an indicator comprising two green fluorescent protein variants linked by a calmodulin binding sequence in living cells. A new class of fluorescent indicators.

CRET
Resonance energy transfer by chemiluminescence (ie, fusion protein with GFP-Aequorin without linking (Aequorea) or with GFP-Obeline).

References Chemiluminescence energy transfer

BRET
Energy transfer due to bioluminescence resonance (ie, interaction between GFP and luciferase (Jellyfish Renilla)).

References Bioluminescence Resonance Energy Transfer (BRET) System: Application to interacting biological clock proteins.

References The following references are cited herein. The entire disclosure of each of these references and each other cited herein is relied upon and incorporated herein.

US patent documents:
No. 6,662,039 B2. Optical probing of neural connections using fluorescent indicators. December 2003, Yuste et al.

FIG. 5 shows the localization of various GFP-aequorin reporters targeted to specific subdomains of presynaptic and posterior compartments. The GFP-Aequorin reporter is targeted to various cellular domains such as the mitochondrial matrix (mtGA) by fusion to synaptotagmin I (SynGA) and to the endoplasmic reticulum (erGA) cavity by fusion to PSD95 (PSDGA). Each low-affinity reporter can selectively detect high-concentration calcium microdomains, indicating a specific cellular activity. Diagram of various GA chimeras for cell specific targeting. The white asterisk indicates the location of the (Asp-119 → Ala) mutation in aequorin, which reduces the Ca ²⁺ binding affinity of the photoprotein as described by Kendall et al., 1992. GA represents GFP-Aequorin without targeting ability, which is called G5A and contains a flexible linker between the two proteins (GA and SynGA are PCT / EP01 / 07057 applications) Published). mtGA is GFP-Aequorin targeted to mitochondria by fusing GA to the excisable targeting sequence of cytochrome c oxidase subunit VIII; erGA is to the N-terminal region of the immunoglobulin heavy chain GFP-Aequorin targeted to the endoplasmic reticulum cavity after fusion; PSDGA is a fusion of GFP-Aequorin to PSD95 for local targeting to the postsynaptic structure. All constructs are under the control of the human cytomegalovirus promoter (pCMV). Ca ²⁺ concentration response curve of mtGA. The recombinant protein is reconstituted with the natural or synthetic analog h-coelenterazine (which is reported to be more sensitive to Ca ²⁺ than the natural complex) after mtGA Ca ²⁺ concentration response curves (pH 7. 2, measured at 26 ° C. (n = 3)). The rate of consumption of aequorin is proportional to [Ca ²⁺ ] in the physiological pCa range. Consumption rate fraction of light the protein comprises an optical radiation in a defined ^[Ca 2+] (L), expressed as the ratio between the maximum light radiation in saturation ^[Ca 2+] (Lmax). Analysis by confocal microscopy of various GFP-Aequorin chimeras targeted to specific subcellular domains. (A) mtGA, GFP-Aequorin is well targeted to the mitochondrial matrix in COS7 and cortical neurons. (B) erGA, GFP-Aequorin is well targeted to the endoplasmic reticulum cavity. (C) GFP-Aequorin fused to the C-terminus of PSDGA, PSD95 protein labeled the Ca ²⁺ -reporter in a punctiform manner, similar to targeting of native proteins in dissociated cortical neurons. (D) SynGA, the synaptic region is labeled with GFP-aequorin fused to the C-terminus of synaptotagmin I, which is a transmembrane protein of synaptic vesicles. Targeted GA reporters are also confirmed by immunohistochemical staining with related antibodies. Cortical cells transfected with a type of GFP-Aequorin that has no targeting ability. GFP-Aequorin was reconstituted with high affinity h-type coelenterazine. (A) GFP fluorescence shows a uniform distribution of Ca ²⁺ reporters. (B, b) Bioluminescence induced by Ca ²⁺ after application of 100 μM NMDA and (C, c) 90 mM KCl to single cortical neurons transfected with GA, and corresponding graph data. (D) A high concentration Ca ²⁺ solution containing digitonin was added at the end of the experiment and the total amount of photoprotein was quantified to calibrate the Ca ²⁺ concentration. Images were obtained at room temperature (23-25 ° C.) using a 40 × objective lens (1.3 NA). Scale reference bar = 15 μm. The change in [Ca ²⁺ ] indicated by the number of detected photons is encrypted with a false color (1-5 photons / pixel), dark blue indicates a small number of pixels, and red indicates a large number of pixels. Ca2 ⁺ influx induced by NMDA in cortical neurons transfected with mtGA. GFP can visualize the Ca ²⁺ reporter expression pattern by fluorescence microscopy as shown in the initial image baseline, where the baseline GFP fluorescence image is overlaid with the pre-stimulated photon image. ing. Using a highly sensitive image photon detector (IPD), bioluminescence induced by Ca ²⁺ was recorded after application of NMDA (100 μM). IPD detection provides a high temporal resolution and a medium spatial resolution. See the zoom region showing a comparison of spatial resolution between (A) CCD fluorescence image, scale bar = 5 μm and (B) IPD photon image. (C) GFP fluorescence image showing the region of interest and corresponding graph data. Scale reference bar = 10 μm. The graph also shows the overall cell response. Each photon image shows light accumulated for 30 seconds. Background <1 photon / second. The color scale shows the emission inflow as 1 to 5 photons / pixel. Bioluminescence activity induced by Ca ²⁺ in cortical neurons transfected with SynGA. In the basal state before the addition of the neuromodulator, the analyzed area showed a high level of activity compared to the background. This is consistently observed in neurons transfected with SynGA. Usually, when GFP-aequorin is regenerated with natural aequorin, the binding affinity of the reporter is low, so it is difficult to detect the resting level of Ca ²⁺ . mtGA and PSDGA generally do not show the same type of activity, but PSDGA sometimes exhibits very localized Ca ²⁺ influx that occurs spontaneously and accidentally. These results suggest that SynGA is targeted to cellular domains that have higher Ca ²⁺ concentrations than are normally reported for cytosolic Ca ²⁺ resting levels. The background photons were less than 1 photon / second in the 256 × 256 pixel area. A 20 × 20 pixel region was selected from various locations along the cell trunk and neurite. The graph data also shows an increase in background count in each region. It is noteworthy that the background is very close to zero and therefore not seen. Ca ²⁺ influx in somatic trunks and neurites after addition of high concentrations of K ⁺ (90 mM KCl). Corresponding (BF) bright field and (FI) fluorescence images, and a superposition of the fluorescence and photon images are shown. Scale reference bar = 20 μm. The photon image was scaled to 1-5 photons / pixel. Cortical neurons transfected with PSDGA. (A) Visualizing GFP fluorescence to identify neurons that show Ca ²⁺ reporter expression, which is similar to that of native PSD95 protein. The photon emission in two dendritic regions (15 × 15 pixels), called D1 and D2, as well as the region of the same size cell trunk was examined and displayed graphically. The kinetics of Ca ²⁺ signaling was found to be identical in the two analyzed dendritic regions, but was found to be significantly different compared to the cell body trunk. (B) Photon image showing the total integral (50-200 seconds) of the photons emitted after the initial application of NMDA. Since all photoproteins in the two dendritic regions analyzed were completely consumed after the first NMDA application, photons were detected only in the soma trunk region after the second NMDA application. The false color scale indicates 1-5 photons / pixel. Scale reference bar = 10 μm. Observation of spontaneous activity recorded from cortical cells expressing PSDGA (GFP-Aequorin fused to PSD95). The response is recorded and displayed graphically under basic conditions (colors represent data collected from the same 20 × 20 pixel area, 1 pixel = 0.65 μm). A hands-on explanation of the corresponding example is made, which includes integrated photon images and graph data. Bioluminescence imaging of long-term Ca ²⁺ kinetics in organotypic hippocampal slice cultures of neonatal mouse brain infected with adenovirus-GFP-aequorin vector. (A) GFP fluorescence shows individual cells expressing the Ca ²⁺ reporter. Activity was recorded for approximately 9 hours until cell death, as evidenced by a large increase in bioluminescent activity and loss of fluorescence, became apparent. Fluorescence images were taken periodically (every 30 minutes) while taking. A representative photon image and corresponding graph data (last 7 hours) are shown. Background <1 photon / second × 10. Map of PSDGA vector. Coding sequence (and corresponding protein sequence) of the insert including PSD95 (nucleotide positions 616-2788), adapter (upper case), GFP (2842-3555), linker (3556-3705, upper case), and aequorin (3706-4275) ). Map of mtGA vector. Insertion fragment containing cytochrome C oxidase subunit VIII excisable targeting sequence (nucleotide positions 636 to 722, upper case), GFP (741 to 1454), linker (1455-1604, upper case) and aequorin (1605 to 2174) Coding sequence (and corresponding protein sequence). Detection of dynamic activity in single cells when GFP-Aequorin is localized to a specific cellular domain. Bioluminescence induced by Ca ²⁺ in cortical neurons transfected with PSDGA. The propagation and response profile of the Ca ²⁺ waves that occur subcellularly have been shown to be very complex. The IPD camera used in these studies provides a resolution on the microsecond timeline, and the integration time is specified only for online visualization. Working on a highly variable time scale makes it possible to fully examine the spatio-temporal properties of Ca ²⁺ activity, which is itself a physiological parameter. Scale reference bar = 20 μm. Ca ²⁺ oscillations induced by electricity in hippocampal neurons. Fluorescence (FI) and bright field image (BF) of a 24-hour culture, with a patch-like pipette (7-10 MΩ) connected to a pulse generator and the body area of the “lower” cell shown in the image Gently contacted. Scale reference bar = 20 μm. The light emission (A) induced by a single 2 millisecond electrical pulse is shown in the graphs A-E shown in the lower panel at 5 second intervals. The applied voltage is described in each graph (the electrode means the battery side to which the pipette is connected). Superimpose the photon image with the bright field image. The light emission fractions (L / Lmax) of the three regions shown in BF are shown in graphs A to E and correspond to continuous electrical stimulation. (F) All Ca ²⁺ transients recorded in each area of interest during a 20 second time period are shown as a function of time. An asterisk represents the first of a series of spontaneous transients. (G) Electrical layout. I. S. Isolated stimulator, R, electrical repeater, A, patch amplifier head. Neurons were transfected with a viral vector containing the GFP-Aequorin gene (see method for more details). GFP fluorescence of 10-day and 5-day-old transgenic and wild-type embryos. Chimeric mtGA (pCAG-Lox-stop-Lox-mtGA) mice were crossed with PGK-CRE mice to activate transgene expression in all cells of the organism from the start of development. (A) Bright field images of 10- and 5-day-old embryos of wild type and transgenic mice. (B) Corresponding GFP fluorescence image. There are no phenotypic abnormalities. GFP fluorescence in neonatal transgenic mice expressing GFP-aequorin protein targeted to mitochondria in all cells. Bright field images and corresponding GFP fluorescence images of mtGA mice after activation of the transgene (by crossing with PGK-CRE) in all cells of the organism from the start of development. (A) paw, (B) upper body back view, (C1) back view of head, and (C2) mtGA targeting in cortex of P1 mtGA mice. There are no physical or behavioral abnormalities in newborns or adult mice. GFP fluorescence images of organs cut out from transgenic newborn mice and organs of wild type mice. GFP fluorescence image of P5 transgenic mtGA and wild type mice. Images of major organs are taken and show strong expression levels in all organs. The highest expression levels are found in the heart and liver. When the transgene is activated at the start of development in all cells, no organ abnormalities are observed. Confocal analysis of mtGA in the cortex of transgenic animals expressing the transgene in all cells. In order to activate mtGA transgene expression in all cells of live animals, transgenic mice were crossed with PGK-CRE mice. Organotypic sections were prepared from P4 mice and kept in culture for 4 days, after which experiments were performed to detect bioluminescence induced by Ca ²⁺ . At the completion of the experiment, sections were fixed and then stained with GFAP (for detection of glial cells) and NeuN (for detection of neurons). Both antibodies were both localized at the location of expression of the mtGA transgene. After fusion to a cytochrome c signal peptide for targeting to the mitochondrial matrix (mtGA), GFP fluorescence shows the expected expression pattern of the GA reporter. These results indicate that the mtGA transgene is expressed in all cells of the brain when transgenic mtGA reporter mice are crossed with PGK-CRE mice that activate Cre in whole cells. Synchronous oscillation of mitochondrial Ca ²⁺ transients in somatosensory cortex of immature mouse brain. (BF, bright field, FI, fluorescence image) Organotype sections (cortex) were excised from P4 transgenic mice expressing GFP-Aequorin targeted to mitochondria in all cells of the brain. Sections were kept in culture for 4-5 days before imaging. After incubation with coelenterazine (wt), sections were perfused in buffer (with or without Mg ²⁺ ). The results of two sections are presented, according to which removal of Mg ²⁺ from the buffer results in Ca ²⁺ oscillations that are detected in the mitochondrial matrix and are completely eliminated by the NMDA antagonist D-APV (50 μM). And is shown to be reversibly blocked. Photons were collected from the 550 μm ² region corresponding to the somatosensory cortex of the I-III / IV and V layers of the brain cortex. Bioluminescence imaging of whole animal of P1 mice with GFP-Aequorin targeted to mitochondria. The left hand mouse is a wild type mouse and the right hand mouse is a transgenic mouse expressing GFP-Aequorin targeted to mitochondria in all cells. Both mice were injected intraperitoneally with coelenterazine (4 μg / g). A to C were separate series and acquired continuous images over time. Gray scale photographs of mice were first collected in the chamber under diode illumination emitting a faint light, and then false color emission images were acquired and overlaid. Each frame shows the light accumulation for 5 seconds. Color reference bars corresponding to light intensities from purple (lowest intensity) to red (highest intensity) are listed at the end of each series. Scale reference bar = 2 cm. Bioluminescence imaging of whole animal of P3 mice with GFP-Aequorin targeted to mitochondria. The left hand mouse is a wild type mouse and the right hand mouse is a transgenic mouse expressing GFP-Aequorin targeted to mitochondria in all cells. Both mice were injected intraperitoneally with coelenterazine (4 μg / g). A and B showed separate sequences and acquired sequential images over time. Gray scale photographs of mice were first collected in the chamber under diode illumination emitting a faint light, and then false color emission images were acquired and overlaid. Each frame shows the light accumulation for 5 seconds. Color reference bars corresponding to light intensities from purple (lowest intensity) to red (highest intensity) are listed at the end of each series. Scale reference bar = 4 cm. Bioluminescence imaging of mitochondrial Ca ²⁺ dynamics in whole animals using higher temporal resolution. The left hand mouse is a wild type mouse and the right hand mouse is a transgenic mouse expressing GFP-Aequorin targeted to mitochondria in all cells. Both mice were injected intraperitoneally with coelenterazine (4 μg / g). An exposure time of 2-5 seconds is shown in each continuous frame. Bioluminescence imaging of mitochondrial Ca ²⁺ dynamics in whole animals using higher temporal resolution. The left hand mouse is a wild type mouse and the right hand mouse is a transgenic mouse expressing GFP-Aequorin targeted to mitochondria in all cells. The image shows the light accumulation for 1 second. The color bar corresponds to the light intensity. Images were acquired using a Xenogen IVIS 100 animal whole body bioluminescence system. Binning = 16, F / stop = 1.

Claims (78)

A method for optical detection of Ca ²⁺ kinetics in a biological system, said method comprising emitting a recombinant Ca ²⁺ sensitive polypeptide comprising or consisting of a chemiluminescent protein linked to a fluorescent protein present in said biological system. Monitoring said photons. 2. The method of claim 1 for optical detection of intracellular Ca2 ⁺ signaling. The method of claim 1 for optically detecting the propagation of a Ca ²⁺ signal to detect transmission from one cell to another.   The method according to any one of claims 1 to 3, wherein the detection is performed in vivo.   The method according to claim 1, wherein the detection is performed ex vivo.   The method according to claim 1, wherein the chemiluminescent protein binds to calcium ions.   The method according to any one of claims 1 to 6, wherein the chemiluminescent protein is aequorin.   8. The method of claim 7, wherein the aequorin is a mutant aequorin having a low affinity for calcium ions.   9. The method of claim 8, wherein the mutant aequorin has the following substitution Asp407 → Ala.   The method according to any one of claims 1 to 9, wherein the fluorescent protein is GFP.   The method according to any one of claims 1 to 9, wherein the fluorescent protein is a GFP variant that emits light at different wavelengths.   12. The method of claim 11, wherein the GFP variant is selected from the group consisting of CFP, YFP, and RFP.   10. The method according to any one of claims 1 to 9, wherein the fluorescent protein is a variant of GFP that improves the photon emission intensity and / or stability of the recombinant polypeptide.   The recombinant polypeptide comprises or consists of aequorin and GFP or a respective variant or variant thereof, wherein the aequorin and GFP are linked by a linker that allows chemiluminescence resonance energy transfer (CRET); The method according to any one of claims 1 to 13.   15. A method according to claim 14, wherein the linker comprises 4 to 63 amino acids, preferably 14 to 50 amino acids. The linker comprises or consists of the sequence [Gly-Gly-Ser-Gly-Ser-Gly-Gly-Gln-Ser] _n , where n is 1-5, preferably n is 1. The method according to claim 14, wherein n is 5.   17. The recombinant polypeptide of any one of claims 1-16, wherein the recombinant polypeptide further comprises a peptide fragment capable of targeting the resulting bioluminescent protein to a specific cellular domain or compartment. Method.   18. The method of claim 17, wherein the peptide fragment is selected from the group comprising synaptotagmin, PSD95, cytochrome C oxidase subunit VIII, immunoglobulin heavy chain.   The method according to any one of claims 1 to 18, wherein the biological system is selected from a cell or a group of cells.   The method according to claim 1, wherein the biological system is a tissue or a part thereof.   The method according to any one of claims 1 to 18, wherein the biological system is a whole animal or a plant.   23. The method of any one of claims 19-21, comprising administering coelenterazine to activate aequorin prior to monitoring photon emission.   23. A method according to any one of claims 19 to 22, comprising administering the recombinant polypeptide to a biological system prior to monitoring photon emission.   23. A method according to any one of claims 21 to 22, wherein the recombinant polypeptide is injected into a biological system, preferably intravenously, intraperitoneally or intramuscularly.   23. The method of any one of claims 19-22, wherein the nucleic acid encoding the recombinant polypeptide is introduced into a biological system.   26. The method of claim 25, wherein the nucleic acid encoding the recombinant polypeptide is delivered to a biological system by a recombinant vector, in particular by a recombinant viral vector.   26. The method of claim 25, wherein the nucleic acid encoding the recombinant polypeptide is introduced into a biological system by gene transfer.   28. The method of any one of claims 21 to 27, wherein the optical detection is performed in a non-human transgenic animal or transgenic plant expressing the recombinant polypeptide from the genome.   30. The method of claim 28, wherein the non-human transgenic animal is a mammal.   30. The method of claim 29, wherein the non-human transgenic animal is a mouse.   31. The method of any one of claims 27-30, wherein expression and / or localization of the recombinant polypeptide is limited to a particular tissue, single cell type, or cell compartment or domain.   32. The method of claim 31, wherein the single cell type is a neural cell, heart cell, or liver cell.   32. The method of claim 31, wherein the cell compartment is mitochondrion or chloroplast. a) characterization of cell group, tissue, cell, or cell compartment or domain development, morphology, or function by GFP fluorescence detection, and b) by the method of any one of claims 1-33, A method of identifying physiological and / or pathological processes, including characterization of Ca ²⁺ kinetics in a group of cells, the tissue, the cells, or the cell compartment or domain. A transgenic non-human animal expressing a recombinant polypeptide sensitive to calcium concentration comprising or consisting of a chemiluminescent protein linked to a fluorescent protein under conditions allowing monitoring of Ca ²⁺ kinetics in vivo.   36. The transgenic non-human animal of claim 35, wherein the chemiluminescent protein binds to calcium ions.   The transgenic non-human animal according to any one of claims 35 to 36, which is capable of realizing non-invasive monitoring in vivo.   38. The transgenic non-human animal according to any one of claims 35 to 37, wherein the recombinant polypeptide is genetically encoded in the animal.   39. The transgenic non-human animal according to any one of claims 35 to 38, wherein the recombinant polypeptide sensitive to calcium concentration is encoded by a polynucleotide inserted in the genome of the transgenic animal.   40. The transgenic non-human animal of claim 39, wherein the polynucleotide encoding the recombinant polypeptide sensitive to calcium concentration is placed under the control of an appropriate transcriptional and / or translational system.   41. The transgenic non-human animal according to any one of claims 35 to 40, wherein the expression and / or localization of the recombinant polypeptide is limited to a specific tissue.   41. The transgenic non-human animal according to any one of claims 35 to 40, wherein the expression and / or localization of the recombinant polypeptide is limited to a single cell type.   43. The transgenic non-human animal according to claim 42, wherein the single cell type is a nerve cell, heart cell, or liver cell.   41. The transgenic non-human animal according to any one of claims 35 to 40, wherein the expression and / or localization of the recombinant polypeptide is limited to a particular cell compartment or domain.   45. The transgenic non-human animal of claim 44, wherein the cell compartment is mitochondria.   46. The transgenic non-human animal according to any one of claims 40 to 45, wherein expression of the polynucleotide encoding the recombinant polypeptide sensitive to calcium concentration is conditional.   47. The method of any one of claims 40 to 46, wherein the polynucleotide encoding the recombinant polypeptide sensitive to calcium concentration is placed under the control of a β-actin promoter and a Lox-stop-Lox sequence. The transgenic non-human animal described.   48. The transgenic non-human animal according to any one of claims 35 to 47, wherein the chemiluminescent protein is aequorin.   49. The transgenic non-human animal according to claim 48, wherein the aequorin is a mutant aequorin having a high affinity for calcium ions.   The transgenic non-human animal according to claim 49, wherein the aequorin is a mutant aequorin Asp407 → Ala.   51. The transgenic non-human animal according to any one of claims 35 to 50, wherein the fluorescent protein is GFP.   52. The transgenic non-human animal according to any one of claims 35 to 51, wherein the fluorescent protein is a GFP variant that emits light at different wavelengths.   53. The transgenic non-human animal of claim 52, wherein said GFP variant is selected from the group consisting of CFP, YFP, and RFP.   52. The transgenic non-human animal according to any one of claims 35 to 51, wherein the fluorescent protein is a mutant of GFP that improves the photon emission intensity or stability of the recombinant polypeptide.   The recombinant polypeptide comprises or consists of aequorin and GFP or their respective variants or variants, the aequorin and GFP being linked by a linker that allows chemiluminescence resonance energy transfer (CRET); The transgenic non-human animal according to any one of claims 35 to 54.   56. The transgenic non-human animal according to claim 55, wherein the linker comprises 4 to 63 amino acids, preferably 14 to 50 amino acids. The linker comprises or consists of the sequence [Gly-Gly-Ser-Gly-Ser-Gly-Gly-Gln-Ser] _n , where n is 1-5, preferably n is 1. Or a transgenic non-human animal according to any one of claims 55 to 56, wherein n is 5.   58. The trans of any one of claims 35 to 57, wherein the recombinant polypeptide further comprises a peptide fragment capable of targeting the resulting bioluminescent protein to a particular compartment or cellular domain. A transgenic non-human animal.   59. The transgenic non-human animal of claim 58, wherein the peptide fragment is selected from the group comprising synaptotagmin, PSD95, cytochrome C oxidase subunit VIII, immunoglobulin heavy chain.   60. The transgenic non-human animal according to any one of claims 35 to 59, wherein the animal is a mammal.   61. The transgenic non-human animal according to any one of claims 35 to 60, wherein the animal is a mouse.   The transgenic progeny of the transgenic non-human animal according to any one of claims 35 to 61.   The transgenic animal which can be obtained by crossing the animal of any one of Claims 45-61 with the mutant animal, especially the animal model which has a known pathological condition. a) introducing a DNA construct into embryonic stem cells of a non-human animal, wherein said DNA construct comprises a sequence encoding a recombinant polypeptide sensitive to calcium concentration, a so-called transgene or The recombinant polypeptide comprises or consists of a chemiluminescent protein linked to a fluorescent protein, the transgene being placed under the control of a promoter and optionally a conditional expression sequence,
b) selecting a positive clone in which the DNA construct has been inserted into the genome of the embryonic stem cell;
35. c) injecting the positive clone into a blastocyst and recovering the chimeric blastocyst, and d) growing the chimeric blastocyst to obtain a non-human transgenic animal. 60. A method for producing a transgenic non-human animal according to any one of 60. a) producing a first transgenic non-human animal by the method of claim 63;
b) The obtained first transgenic non-human animal expresses an endonuclease that acts on the conditional expression sequence in a tissue or cell in which the expression of the recombinant polypeptide is required. Crossing with animals,
62. Production of a transgenic non-human animal according to any one of claims 46 to 61, comprising c) recovering the transgenic non-human animal expressing the recombinant polypeptide in a specific tissue or cell. Law.   66. The method of claim 65, wherein the conditional expression sequence is a Lox site and the endonuclease is Cre.   67. The method of any one of claims 65 to 66, wherein the polynucleotide encoding the endonuclease is placed under the control of a cell specific promoter.   68. The method of any one of claims 65 to 67, wherein the cell specific promoter is active in the liver, heart, or brain.   69. The DNA construct of any one of claims 65-68, wherein the DNA construct comprises or consists of a promoter, a conditional expression sequence, and a transgene encoding the recombinant polypeptide that is sensitive to calcium concentration. the method of.   70. Any of claims 65-69, wherein the DNA construct comprises or consists of a transgene encoding the recombinant polypeptide that is sensitive to a β-actin promoter, a Lox-stop-Lox sequence, and calcium concentration. The method according to claim 1.   The method according to any one of claims 65 to 70, wherein the DNA construct is inserted into the genome of the embryonic stem cell by homologous recombination.   72. The method of any one of claims 65-71, wherein the DNA construct is inserted into a reconstituted HPRT locus.   73. The method according to any one of claims 64-72, wherein the chemiluminescent protein is aequorin.   74. The method of any one of claims 64-73, wherein the fluorescent protein is GFP.   75. The recombinant polypeptide according to any of claims 64-74, wherein the recombinant polypeptide comprises or consists of aequorin and GFP, wherein the aequorin and GFP are linked by a linker that allows chemiluminescence resonance energy transfer (CRET). 2. The method according to item 1.   76. The method of any one of claims 64-75, wherein the linker comprises 4 to 63 amino acids, preferably 14 to 50 amino acids. The linker comprises or consists of the sequence [Gly-Gly-Ser-Gly-Ser-Gly-Gly-Gln-Ser] _n , where n is 1-5, preferably n is 1. 77. The method of any one of claims 64-76, wherein n is 5. a) In a transgenic animal expressing a recombinant polypeptide sensitive to the calcium concentration according to any one of claims 35 to 61, by the method according to any one of claims 1 to 33, detecting the kinetics of Ca ^2+, optionally quantifying the ^{Ca 2+} that,
b) administering or expressing in the transgenic animal the molecule of interest;
c) repeating step a);
d) the injection position of the front and rear of the ^{Ca 2+,} kinetics, optionally comparing the amount, where the position of ^{Ca 2+,} kinetics, and / or the amount of change, suggesting that the molecule can modulate the ^{Ca 2+} Yes,
62. A method for screening a molecule capable of modulating Ca2 ⁺ in a transgenic non-human animal according to any one of claims 35 to 61, comprising:

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